Point-of-Care Platform for Diagnosis of Venous Thrombosis by Simultaneous Detection of Thrombin Generation and D-Dimer in Human Plasma.

Venous thromboembolism (VTE) refers to a blood clot that starts in a vein. The risk of developing VTE is highest after major surgery or a major injury, or when someone has heart failure, cancer, or infectious disease (e.g., COVID-19). Without prompt treatment to break up clots and prevent more from forming, VTE can restrict or block blood flow and oxygen, which can damage the body tissue or organs. VTE can occur without any obvious signs, and imaging technologies are used. Alternatively rapid measurement of thrombin generation (TG) and D-dimer could be used to make a fast, portable, and easy-to-use diagnostic platform for VTE. Here, we have demonstrated a diagnostic sensing platform with the ability of simultaneous detection of TG and D-dimer in human plasma. Modifications were made to both the assay protocols to eliminate the need for sample dilution and incubation steps. Using a substantially reduced sample volume, the measurement results show comparable performance to the gold standard method. Our platform is able to deliver accurate and cost-effective results for both TG and D-dimer assays when using undiluted plasma in under 15 min. The assays presented are therefore a good candidate technology for use in a point-of-care platform to diagnose VTE.

Haematological variables and risk of future venous thromboembolism in the British Regional Heart Study on men. Combined D-dimer and APTT as a predictive test for thromboembolism?

We examined the associations between haematological and inflammatory variables with future venous thromboembolism (VTE), in 3494 men aged 60-79 years, with no previous history of VTE or myocardial infarction, who were not receiving oral anticoagulants. After a mean follow-up period of 18 years, there were 149 confirmed cases of fatal or non-fatal VTE (deep vein thrombosis and/or pulmonary embolism). Among classical cardiovascular risk factors, only obesity and cigarette smoking were associated with VTE risk. After adjustment for age, obesity and smoking, VTE risk was associated with coagulation factor VIII, factor IX, von Willebrand factor (VWF), activated partial thromboplastin time (APTT), and fibrin D-dimer. Hazard ratios (95% CI) for top to bottom quarters (bottom to top for APTT), were respectively 2.17 (1.37, 3.44), 2.15 (1.30, 3.53), 2.02 (1.27, 3.22), 2.43 (1.47, 4.02) and 3.62 (2.18, 6.08). The 11% of men with both the shortest APTT and highest D-dimer combined had a 5.02 (2.37, 10.62) higher risk of VTE. VTE risk was not associated with fibrinogen, factor VII or activated protein C resistance; full blood count variables or with inflammatory markers, plasma viscosity, C-reactive protein or interleukin-6. The combination of D-dimer and APTT merits evaluation as an adjunct to VTE risk prediction scores.

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region.

OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.

Association of D-dimer with long-term prognosis in type 2 diabetes mellitus patients with acute coronary syndrome.

BACKGROUND AND AIMS: Type 2 diabetes mellitus (DM) accounts for more and more individuals worldwide. D-dimer has been demonstrated to be associated with cardiovascular diseases. The aim is to study the potential impact of D-dimer on the long-term prognosis of acute coronary syndrome (ACS) in the special population with type 2 DM. METHODS AND RESULTS: A total of 2265 consecutive patients with DM and ACS were eligible in the study. Patients were divided into four groups according to quartiles of D-dimer concentration. Univariate and multivariate Cox regression analysis were conducted to explore the prognostic value of D-dimer for future outcomes. Patients with higher level of D-dimer presented with higher percentage of major adverse cardiovascular events (MACEs) (23.7%), all-cause death (18.3%) and cardiovascular (CV) death (9.4%) in Quartile 4. In multivariate Cox regression analysis, D-dimer was demonstrated to be independently associated with MACEs, all-cause death and CV death. The prognostic value of D-dimer is still significant in subgroups of HbA1C <7% and ≥7%. In Kaplan-Meier analysis, higher D-dimer showed poorer prognosis in MACEs, all-cause death and CV death (all log rank p < 0.001). The area under the curve (AUC) by receiver operating characteristic (ROC) curve analysis is 0.609 for MACEs, 0.708 for all-cause death, 0.747 for CV death (p < 0.001). CONCLUSION: The present study demonstrated the independent predictive value of D-dimer for outcomes in DM patients with ACS. In addition, for the first time, we explored the prognostic value in different glucose control status.

Endothelial Dysfunction in the Brain: Setting the Stage for Stroke and Other Cerebrovascular Complications of COVID-19.

The Coronavirus disease 2019 (COVID)-19 pandemic has already affected millions worldwide, with a current mortality rate of 2.2%. While it is well-established that severe acute respiratory syndrome-coronavirus-2 causes upper and lower respiratory tract infections, a number of neurological sequelae have now been reported in a large proportion of cases. Additionally, the disease causes arterial and venous thromboses including pulmonary embolism, myocardial infarction, and a significant number of cerebrovascular complications. The increasing incidence of large vessel ischemic strokes as well as intracranial hemorrhages, frequently in younger individuals, and associated with increased morbidity and mortality, has raised questions as to why the brain is a major target of the disease. COVID-19 is characterized by hypercoagulability with alterations in hemostatic markers including high D-dimer levels, which are a prognosticator of poor outcome. Together with findings of fibrin-rich microthrombi, widespread extracellular fibrin deposition in affected various organs and hypercytokinemia, this suggests that COVID-19 is more than a pulmonary viral infection. Evidently, COVID-19 is a thrombo-inflammatory disease. Endothelial cells that constitute the lining of blood vessels are the primary targets of a thrombo-inflammatory response, and severe acute respiratory syndrome coronavirus 2 also directly infects endothelial cells through the ACE2 (angiotensin-converting enzyme 2) receptor. Being highly heterogeneous in their structure and function, differences in the endothelial cells may govern the susceptibility of organs to COVID-19. Here, we have explored how the unique characteristics of the cerebral endothelium may be the underlying reason for the increased rates of cerebrovascular pathology associated with COVID-19.

Mass spectrometry-based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots.

The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the αC region of fibrinogen α. Our findings expand the known native cross-linked species from one to over 100 and suggest distinct antiparallel registries for interprotofibril association and covalent attachment of serpins that regulate clot dissolution.

Evaluation of D-dimer levels in aqueous humor of rabbit eyes with and without induced intraocular fibrin and fibrinolytic treatment.

OBJECTIVE: To analyze D-dimer concentrations in aqueous humor (AH) of rabbit eyes under physiological conditions, after induction of fibrin clots, and following fibrinolytic therapy. ANIMALS STUDIED: Prospective study measuring D-dimers in aqueous humor of rabbit eyes with induced fibrin clots (n = 44). PROCEDURES: Rabbits were purchased in two groups, which led to two temporally separated experimentation groups. Different treatment protocols were compared for their efficacy in fibrin reduction (slit-lamp examination, high-resolution ultrasound). AH was taken from left eyes before clot induction (baseline, day 1), 24 hours later after clot establishment/prior to drug administration (post-induction, day 2) and 48 hours after clot induction (post-treatment, day 3). An enzyme-linked immunosorbent assay (ELISA) was performed to measure intraocular D-dimer concentrations RESULTS: D-dimer concentrations were measurable in all samples. There were no differences in D-dimer levels across time points or treatments within the arrival groups. However, a significant difference in mean D-dimer levels was observed between the two arrival groups (group 1:3.1 µg/mL; group 2:6.1 µg/mL; P < .0001), which made a direct comparison of treatment groups impossible. Clinically, all eyes displayed fibrin clots in the anterior chamber and different treatment types led to significant differences in clot resolution (clot size reduction after intracameral treatment: 98%, topical treatment: 60%, no treatment: 40%). CONCLUSION: D-dimers were identified in all AH samples of rabbits with large variability between samples. D-dimer levels were neither predictive for differences in induced fibrin formation nor for drug efficacy.

What makes D-dimer assays suspicious-heterophilic antibodies?

BACKGROUND: Heterophilic antibodies are still an important source of interference in immunoassays, but reports of interference with D-dimers are rare. Are D-dimer level abnormalities, found in the clinic, caused by heterophilic antibodies as well, or are other mechanisms involved? We will elaborate on this issue through two different examples in this article. METHODS: Serum from two patients with significantly elevated levels of D-dimers were measured and compared by different methods, diluted, and dealt with heterophilic antibody blockers. At the same time, to retrieve the interference, we focused on the cause of D-dimer false positives and made a systematic review of the literature. RESULTS: The D-dimer values were normal (0.49 and 0.15 µg/mL) detected with different testing method and decreased after addition of heterophilic antibody blocking reagent. According to literature data, there were 66.7% (4/6) references showed the interference were heterophilic antibody. CONCLUSIONS: The influence of heterophilic antibodies on the measurement of D-dimers remains a big challenge. Different measuring instruments and methods may have significant differences in the measurement of D-dimers. By using a combination of instrumental methods for measuring, incorporating heterophilic antibody blockers, and combining with clinical performance and imaging data, most of the interference can be eliminated.

Plasmin-driven fibrinolysis in a quasi-two-dimensional nanoscale fibrin matrix.

Fibrin plays a fundamentally important role during hemostasis. To withstand the shear forces of blood flow and prevent embolisation, fibrin monomers form a three-dimensional polymer network that serves as an elastic scaffold for the blood clot. The complex spatial hierarchy of the fibrin meshwork, however, severely complicates the exploration of structural features, mechanical properties and molecular changes associated with the individual fibers of the clot. Here we developed a quasi-two-dimensional nanoscale fibrin matrix that enables the investigation of fibrin properties by topographical analysis using atomic force microscopy. The average thickness of the matrix was ∼50 nm, and structural features of component fibers were accessible. The matrix could be lysed with plasmin following rehydration. By following the topology of the matrix during lysis, we were able to uncover the molecular mechanisms of the process. Fibers became flexible but retained axial continuity for an extended time period, indicating that lateral interactions between protofibrils are disrupted first, but the axial interactions remain stable. Nearby fibers often fused into bundles, pointing at the presence of a cohesional force between them. Axial fiber fragmentation rapidly took place in the final step. Conceivably, the persisting axial integrity and cohesion of the fibrils assist to maintain global clot structure, to prevent microembolism, and to generate a high local plasmin concentration for the rapid, final axial fibril fragmentation. The nanoscale fibrin matrix developed and tested here provides a unique insight into the molecular mechanisms behind the structural and mechanical features of fibrin and its proteolytic degradation.

Biochemical and structural analysis of the interaction between ß-amyloid and fibrinogen.

The majority of patients with Alzheimer disease (AD) suffer from impaired cerebral circulation. Accumulating evidence suggests that fibrinogen, the main protein component of blood clots, plays an important role in this circulatory dysfunction in AD. Fibrinogen interacts with ß-amyloid (Aß), forming plasmin-resistant abnormal blood clots, and increased fibrin deposition is found in the brains of AD patients and mouse models. In this study, we investigated the biochemical and structural details of the Aß-fibrinogen interaction. We identified the central region of Aß42 as the most critical region for the interaction, which can be inhibited by specific antibodies against the central region of Aß and by naturally occurring p3 peptides, Aß17-40 and Aß17-42. X-ray crystallographic analysis revealed that Aß42 binding to fragment D of fibrinogen induced a structural change in the C-terminal region of the fibrinogen ß-chain (ß384-393). Furthermore, we identified an additional Aß-binding site within the αC region of fibrinogen. Aß binding to this αC region blocked plasmin-mediated fibrin cleavage at this site, resulting in the generation of increased levels of a plasmin-resistant fibrin degradation fragment. Overall, our study elucidates the Aß-fibrinogen interaction and clarifies the mechanism by which Aß-fibrinogen binding delays fibrinolysis by plasmin. These results may facilitate the development of effective therapeutics against the Aß-fibrinogen interaction to treat cerebrovascular abnormalities in AD.

Orthotopic liver transplant for multifocal lymphangioendotheliomatosis with thrombocytopenia.

An eight-yr-old female with a history of multifocal lymphangioendotheliomatosis and thrombocytopenia presented for MVT. The patient had multiple vascular lesions in the skin and stomach in infancy. Although her cutaneous lesions resolved with vincristine and methylprednisolone, her gastric lesions persisted. Eight yr later, she was diagnosed with portal hypertension and decompensating liver function despite therapy with bevacizumab, propranolol, furosemide, and spironolactone. Upon presentation, she was found to have a Kasabach-Merritt-like coagulopathy in association with multiple lesions in her GI tract and persistent gastric lesions. Although treatment with methylprednisolone and sirolimus normalized her coagulation factors and d-dimer levels, she never developed sustained improvement in her thrombocytopenia. Her liver function continued to deteriorate and she developed hepatorenal syndrome. Given better outcomes after OLT in comparison with MVT, she underwent OLT, with the plan to manage her GI lesions with APC post-transplant. Post-transplant, her liver function and coagulopathy normalized, and GI tract lesions disappeared upon screening with capsule endoscopy. The patient is doing well, without recurrence of either GI lesions or thrombocytopenia, at 18 months after transplantation.

[Clinical features of liver cirrhosis complicated by portal vein thrombosis and related risk factors].

Objective: To investigate the clinical features of patients with liver cirrhosis complicated by portal vein thrombosis (PVT) and related risk factors. Methods: A total of 65 patients with liver cirrhosis complicated by PVT who were diagnosed and treated from June 2013 to June 2015 were enrolled as PVT group, and 70 cirrhotic patients without PVT were enrolled as controls (non-PVT group). The data collected included general information, results of laboratory examination, imaging findings, clinical manifestations, and complications. The clinical features were compared between the two groups, and related risk factors were screened out. Results: There were no significant differences between the PVT group and non-PVT group in age, sex, nation, etiology, white blood cell count, platelet count, international normalized ratio, activated partial thromboplastin time, fibrinogen, serum creatinine, total bilirubin, and the diameter of the splenic vein (all P > 0.05), while between these two groups, there were significant differences in D-dimer (1.87±1.45 mg/ml vs 0.55±0.58 mg/ml, P < 0.05), fibrinogen degradation product (FDP) level (18.57±19.46 µg/ml vs 5.45±6.00 µg/ml, P < 0.05), hemoglobin (99.32±26.73 g/L vs 112.64±25.03 g/L, P < 0.05), albumin (28.51±5.19 g/L vs 33.07±7.94 g/L, P < 0.05), the diameter of the portal vein (12.53±2.70 mm vs 11.17±1.79 mm, P < 0.05), spleen thickness (5.12±0.95 cm vs 4.56±0.83 cm, P < 0.05), spleen length (15.35±3.21 cm vs 13.86±2.82 cm, P < 0.05), and Child-Pugh score (7.66±2.06 vs 6.93±1.87, P < 0.05). The two groups showed no significant differences in diarrhea, ileus, hepatorenal syndrome, and hepatic encephalopathy (P > 0.05), but showed significant differences in abdominal pain (18 vs 7 cases, P < 0.05), fever (17 vs 4 cases, P < 0.05), esophageal variceal bleeding (22 vs 9 cases, P < 0.05), and spontaneous peritonitis (24 vs 12 cases, P < 0.05). D-dimer (OR = 4.290, P < 0.000) and mean platelet volume (OR = 1.294, P = 0.023) were independent risk factors for PVT in patients with liver cirrhosis. Conclusion: Cirrhotic patients with a high degree of liver cirrhosis, high levels of D-dimer and FDP, and a large diameter of the portal vein tend to have a high incidence rate of PVT. PVT can aggravate the clinical symptoms and significantly increase complications in patients with liver cirrhosis. An increased D-dimer level and a greater width of the main portal vein are independent risk factors for PVT in patients with liver cirrhosis.

[Risk factors of lung cancer complicated with symptomatic venous thromboembolism].

OBJECTIVE: To seek risk factors of VTE in patients with lung cancer through analysis of clinical features of patients with lung cancer complicated with venous thromboembolism (VTE). METHODS: Retrospective investigation was performed on patients diagnosed with lung cancer and with complete clinical data who were hospitalized in Peking University People's Hospital from January 1, 2010 to December 31, 2014. According to the presence of symptomatic VTE, patients were distributed into two groups, VTE group and control group. Patients' clinical data and laboratory parameters were collected. Single factor analysis was applied to compare the differences between the two groups. t test or nonparametric test was applied for intragroup comparison of measurement data, and chi-square test was applied for the comparison of counting information. Logistic regression analysis was applied to explore risk factors of venous thromboembolism. For VTE patients with this diagnosis when they were hospitalized, D-dimer and PT were obtained after the occurrence of VTE, so D-dimer and PT were eliminated in the multiple factors analysis. SPSS 13.0 statistical software was applied for statistical management and analysis. RESULTS: 548 patients with lung cancer were include in the investigation, with male 357, female 191, average age of (63.8±10.9) years old, 46 patients in VTE group and 502 patinets in control group. According to the results of single factor analysis in gender, age, tumor pathologic type, tumor stage, WBC, Hb, PLT, CEA, ALT, FIB, D-dimer, PT, APTT, PT-INR, the tumor stage (χ(2)=14.177), CEA (t=2.129) and Hb (t=-2.424) were risk factors for lung cancer patients complicated with venous thromboembolism. Logistic regression analysis showed that tumor stage was the independent risk factor of lung cancer complicated with venous thromboembolism (OR 2.058, 95%CI 1.307-3.238, P=0.002) , and CEA (r=0.395, P<0.001) and Hb (r=-0.144, P=0.001) were associated with lung cancer stage. The area under the curve formed by D-dimer predicting VTE was 0.825 (95%CI 0.751-0.900, P<0.001). CONCLUSION: Tumor stage is the only risk factor for lung cancer patients complicated with venous thromboembolism in the study. However, because this study is a retrospective study, other potential high risk factors causing VTE cannot be excluded.

[Risk factors for pulmonary embolism in acute exacerbation of chronic obstructive pulmonary disease].

OBJECTIVE: To study the risk factors for acute pulmonary embolism (PE) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). METHODS: From November of 2009 to May of 2014, 522 admitted patients [aged 42-93 years, mean(72±9)] with AECOPD received CT pulmonary angiography(CTPA) in the First Affiliated Hospital of Guangzhou Medical University. The patients were classified as PE positive (positive result on CTPA) or PE negative (negative results on CTPA), and related risk factors for PE were analyzed. RESULTS: The frequency of PE was 10.3% in this series of 522 patients with AECOPD. Single factor analysis showed that the following factors were significantly different (χ(2)=4.32-57.06, mean P<0.05)between PE positive and PE negative groups: age≥70 years, immobilization≥3 days, deep vein thrombosis(DVT) and a history of venous thromboembolism(VTE), cor pulmonale caused by COPD, pneumonia, stroke, artery embolization, atrial fibrillation, lower extremity edema, the levels of N-terminal pro-brain natriuretic peptide(NT-proBNP) and D-dimmer. Multiple regression analysis showed that immobilization ≥3 days(OR=25.36, 95%CI: 7.42-86.69, P<0.001), lower extremity edema(OR=7.34, 95%CI: 3.43-15.71, P<0.001) and D-dimmer≥2 000 µg/L(OR=10.10, 95%CI: 2.25-45.42, P=0.003) were the risk factors. The ratio for purulent sputum was 48.1%(26/54) in the PE positive group, and 42.6% (23/54) of the patients showed concurrent purulent sputum and increase of blood markers of infection. The frequency of purulent sputum between PE positive and PE negative groups was not different. CONCLUSIONS: Patients with AECOPD admitted to hospital should be considered for the presence of PE if they had the risk factors of immobilization≥3 days, lower extremity edema and D-dimmer ≥2 000 µg/L.

Covalent structure of single-stranded fibrin oligomers cross-linked by FXIIIa.

FXIIIa-mediated isopeptide γ-γ bonds are produced between γ polypeptide chains of adjacent monomeric fibrin. Despite the use of the different methodological approaches there are apparently conflicting ideas regarding the orientation of γ-γ bonds. To identify the orientation of these bonds a novel approach has been applied. It was based on self-assembly of soluble cross-linked fibrin protofibrils ongoing in the urea solution of moderate concentrations followed by dissociation of protofibrils in the conditions of increasing urea concentration. The oligomers were composed of monomeric desA fibrin molecules created by cleavage of the fibrinopeptides A from fibrinogen molecules with thrombin-like enzyme, reptilase. The results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated an emergence of the double-stranded rod-like fibrin protofibrils. For the first time, the protofibrils are proved to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures. Since no accumulation of α polymers has been found the covalent structure of soluble single-stranded fibrin oligomers is entirely brought about by γ-γ bonds. The results of this study provide an extra evidence to support the model of the longitudinal γ-γ bonds that form between the γ chains end-to-end within the same strand of a protofibril.

[Forensic Analysis of 33 Cases of Fatal Pulmonary Thromboembolism].

OBJECTIVE: To explore the related risk facts of pulmonary thromboembolism (PTE) and analyze the relation between PTE and the trauma or medical behavior by investigating the cases of PTE. METHODS: Thirty-three cases were selected from Institute of Forensic Science (IFS) from 2000 to 2014. RESULTS: In 33 cases, 16 decedents were male, 17 decedents were female; different degrees of dyspnea, chest tight- ness and syncope symptoms were the clinical manifestation of the deceased; the thrombus was mainly distributed in the left and right pulmonary arteries. The main source of embolism was the deep vein of lower limb and the left probability was higher. Trauma, limited position, operation and cardiovascular disease showed high-risk factors of PTE; D-Dimer test, hemolytic test and computer tomography pul- monary angiography were the diagnostic tools for PTE. In some cases, trauma and medical malpractice could be involved in the cause of death. CONCLUSION: Non-typical clinical symptoms present in the most cases caused by PTE, and these cases always show many high-risk factors. The relation between PTE and injury or medical behavior should be considered carefully in the forensic pathological practice.

Predictive Value of Combined Plasma D-Dimer, SCUBE1, and Right Ventricular Tei Index for the Prognosis of Elderly Patients with Acute Pulmonary Thromboembolism.

Acute pulmonary thromboembolism (APTE) has become a non-negligible clinical concern due to its high mortality and complex symptoms. Early diagnosis and prognostic assessment of APTE are of great significance for the long-term benefits of patients, especially elderly patients. Elderly patients with pulmonary embolism (n = 250) who presented to our hospital from January 2018 to July 2021 were recruited into this study. In addition, 50 healthy elderly people with no history of allergies were selected as the control group. An enzyme-linked immunosorbent assay (ELISA) method was used to determine concentrations of D-dimer and signal peptide-CUB-EGF domain-containing protein-1 (SCUBE1) in their plasma. Right ventricular volume contraction time (ICT), ejection time (ET), and isovolumic relaxation time (IRT) were determined by Doppler ultrasound. Right ventricular Tei index was calculated as (ICT + IRT)/ET. High plasma D-dimer, plasma SCUBE1, and right ventricular Tei index are risk factors for poor prognosis in APTE patients after treatment. Plasma D-dimer, plasma SCUBE1, and right ventricular Tei index have predictive value for poor prognosis in APTE patients. Their combined detection (0.256*DD +0.04*SCUBE1 + 10.188*Tei) can improve the sensitivity and specificity of prediction. There is a predictive value of combined plasma D-dimer, SCUBE1, and right ventricular Tei index for the prognosis of elderly patients with APTE.

Increased plasma D-dimer levels may be a promising indicator for diabetic peripheral neuropathy in type 2 diabetes.

Background: Increased plasma D-dimer levels have been reported to be associated with a range of adverse health outcomes. This study aimed to determine whether plasma D-dimer is connected to diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes (T2D). Methods: This study was part of a series exploring the potential risks for DPN. All patients were questioned for neurologic symptoms, examined for neurologic signs, and received nerve conduction studies to collect nerve action potential onset latency, amplitude, and nerve conduction velocity (NCV). Composite Z scores of latency, amplitude, and NCV were calculated. DPN was confirmed as both at least a neurologic symptom/sign and an abnormality of nerve conduction studies. Coagulation function indices, such as plasma D-dimer levels, were also synchronously detected. Results: We finally recruited 393 eligible patients for this study, of whom 24.7% (n = 97) were determined to have DPN. The plasma D-dimer level was found to be closely associated with the composite Z score of latency, amplitude, and NCV after adjusting for other coagulation function indices and clinical covariates (latency: ß = 0.134, t = 2.299, p = 0.022; amplitude: ß = -0.138, t = -2.286, p = 0.023; NCV: ß = -0.139, t = -2.433, p = 0.016). Moreover, the prevalence of DPN in the first, second, third, and fourth quartiles (Q1, Q2, Q3, and Q4) of the D-dimer level was 15.2%, 15.9%, 26.4%, and 42.7%, respectively (p for trend < 0.001). The corresponding adjusted odds ratios and 95% CIs for DPN in D-dimer quartiles were 1, 0.79 (0.21-2.99), 1.75 (0.49-6.26), and 5.17 (1.38-19.42), respectively. Furthermore, the optimal cutoff value of the plasma D-dimer level to discriminate DPN was ≥0.22 mg/L (sensitivity = 67.01%, specificity = 58.78%, and Youden index = 0.26) after analysis by the receiver operating characteristic curve. Conclusions: Increased plasma D-dimer levels may be a promising indicator for DPN in patients with T2D.

Self-assembly of soluble unlinked and cross-linked fibrin oligomers.

Self-assembly of soluble unlinked and cross-linked fibrin oligomers formed from desA-fibrin monomer under the influence of factor XIIIa was studied in the presence of non-denaturing urea concentrations. By methods of elastic and dynamic light scattering combined with analytical ultracentrifugation, desA-fibrin oligomers formed in both the presence and absence of the factor XIIIa were shown to be ensembles consisting of soluble rod-like double-stranded protofibrils with diverse weight and size. Unlinked and cross-linked soluble double-stranded protofibrils can reach the length of 350-450 nm. The structure of soluble covalently-linked protofibrils is stabilized by isopeptide γ-dimers. Electrophoretic data indicate a complete absence of isopeptide bonds between α-chains of desA-fibrin molecules. The molecular mechanism of formation of soluble rod-like fibrin structures and specific features of its covalent stabilization under the influence of factor XIIIa are discussed.

Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.

BACKGROUND: Thrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 µM) and the splice variant γ'-domain (Kd ~ 0.1 µM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability. METHODS: A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. RESULTS: Large increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction. CONCLUSIONS: TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin. SIGNIFICANCE: The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.