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Angiotensin converting enzyme 2 (ACE2) is an endogenous negative regulator of the renin-angiotensin system, a key factor in the development of cardiovascular disease (CVD). ACE2 is also used by SARS-CoV-2 for host cell entry. Given that COVID-19 is associated with hypercoagulability, it is timely to explore the potential relationship between plasma ACE2 activity and the coagulation profile. In this cross-sectional study, ACE2 activity and global coagulation assays (GCA) including thromboelastography, thrombin, and fibrin generation were measured in adult healthy controls (n = 123; mean age 41 ± 17 years; 35% male) and in patients with cardiovascular risk factors and/or disease (n = 258; mean age 65 ± 14 years; 55% male). ACE2 activity was significantly lower in controls compared to patients with cardiovascular risk factors and/or disease (median 0.10 (0.02, 3.33) vs. 5.99 (1.95, 10.37) pmol/mL/min, p < 0.001). Of the healthy controls, 48% had undetectable ACE2 activity. Controls with detectable ACE2 had lower maximum amplitude (p < 0.001). In patients with cardiovascular risk factors and/or disease, those in the 3rd tertile were older and male (p = 0.002), with a higher Framingham grade and increased number of cardiovascular risk factors (p < 0.001). In conclusion, plasma ACE2 activity is undetectable to very low in young healthy controls with minimal clinically relevant associations to GCA. Patients with cardiovascular risk factors and/or disease have increased plasma ACE2 activity, suggesting that it may be an important biomarker of endothelial dysfunction and atherosclerosis.

Cardiovascular disease remains the leading cause of death in the era of modern medicine despite major advancements in this field. Current available clinical surrogate markers and blood tests do not adequately predict individual risk of cardiovascular disease. A more precise and sophisticated tool that can reliably predict the thrombosis and bleeding risks at an individual level is required in order for clinicians to confidently recommend early interventions with a favorable risk-benefit profile. Critical to the development of this tool is the assessment and understanding of Virchow's triad and its complex interactions between hypercoagulability, endothelial dysfunction and vessel flow, a fundamental concept to the development of thrombosis. This review explores the pathophysiology of cardiovascular disease stemming from the triad of factors and how individualized risk assessment can be improved through the multimodal use of tools such as global coagulation assays, endothelial biomarkers and vessel flow assessment.

Assessing the risk of recurrent venous thromboembolism (VTE), particularly when patients are anticoagulated, remains a major challenge largely due to the lack of biomarkers. Blood was sampled from adult VTE patients recruited between January 2018 and September 2020, while receiving therapeutic anticoagulation. Results were compared to 144 healthy subjects (34.7% male, median age 42 years). Overall haemostatic potential (OHP) assay, a spectrophotometric assay, was performed on platelet-poor plasma, in which fibrin formation (triggered by small amounts of thrombin (overall coagulation potential, OCP)) and fibrinolysis (by the addition of thrombin and tissue plasminogen activator (OHP)) are simultaneously measured. Results were obtained from 196 patients (52.6% male, mean age 57.1 years). Compared to healthy subjects, VTE patients displayed significantly higher OCP (39.6 vs 34.5 units, p < 0.001) and OHP (9.3 vs 6.4 units, p < 0.001) as well as lower overall fibrinolytic potential (75.6 v s81.1%, p < 0.001). All 16 VTE recurrences, including 11 unprovoked, occurred above an OCP cut-off of 40th percentile (recurrence rate 4.32/100 patient-years (100PY), 95% confidence interval (CI) 2.39-7.80, p = 0.002). Of 97 patients who subsequently discontinued anticoagulation, all unprovoked VTE recurrences (n = 9) occurred above the 40th OCP percentile (recurrence rate 9.10/100PY, 95% CI 4.74-17.49, p = 0.005) and the 40th OHP percentile (recurrence rate 8.46/100PY, 95% CI 4.40-16.25, p = 0.009). Our pilot study demonstrates that the OHP assay can detect a hypercoagulable and hypofibrinolytic state in anticoagulated VTE patients and may be able to risk stratify VTE recurrence, allowing for more individualised decision on long-term anticoagulation. Further larger prospective studies are required.

Deep vein thrombosis (DVT) frequently leads to post-thrombotic syndrome (PTS) which is challenging to predict and prevent. Identifying those at high risk of developing PTS may help to focus preventative strategies. Adults were recruited within 3 months of DVT diagnosis. Blood was sampled during the therapeutic anticoagulation phase. Overall hemostatic potential (OHP) assay, a spectrophotometric assay, was performed on platelet-poor plasma (PPP). In this assay, fibrin formation is triggered by small amounts of thrombin and termed the overall coagulation potential (OCP). Simultaneously, thrombin and tissue plasminogen activator are added to PPP and the resulting fibrin aggregation curve is the overall hemostatic potential (OHP). Fibrinolysis is expressed by the parameter overall fibrinolytic potential (OFP%). Patients were followed up at regular intervals. PTS was diagnosed if the Villalta score was ≥5 at least 3 months after the DVT diagnosis. Results were obtained from 190 patients (53.7% male, mean age 56.9 years). PTS developed in 62 (32.6%) patients. Patients with PTS displayed significantly higher median OCP (45.8 vs. 38.8 units, p = 0.010), OHP (12.8 vs. 9.2 units, p = 0.005) and significantly lower OFP (74.1 vs. 75.6%, p = 0.050). PTS patients had higher neutrophil/lymphocyte ratios (NLR) (2.3 vs. 1.9, p = 0.007). After multivariate analysis, proximal DVT location, history of varicose veins, NLR ≥ 2.6, OHP > 13.0 units and weight >108 kg were independent predictors for PTS. The c-statistic of the multivariate model was 0.77. This pilot study suggests that OHP testing while patients are still anticoagulated may assist in the prediction of PTS development and could assist in prognostication and targeting of preventative measures. However, larger prospective studies are needed to confirm these findings.

Abnormal coagulation and fibrinolysis contributes to the respiratory distress syndrome in COVID-19. We aimed to explore the association of impaired fibrinolytic potential with disease severity and oxygen requirement in hospitalized patients. Adults admitted to hospital with confirmed COVID-19 infection between 1-31 January 2022 were included, corresponding to the first Omicron outbreak in Melbourne, Victoria. The first citrated plasma sample requested within 24 h of the patient's presentation was obtained and analyzed by the overall hemostatic potential (OHP) assay, a spectrophotometric assay in which fibrin formation (triggered by small amounts of thrombin (OCP)) and fibrinolysis (by the addition of thrombin and tissue plasminogen activator (OHP and OFP%)) were simultaneously measured. There were 266 patients (median 72 years, 52.9% male), of which 49.6% did not require oxygen therapy. COVID-19 severity and requirement for oxygen was significantly associated with higher OCP, OHP, and lower OFP%. Vaccinated individuals compared with non-vaccinated individuals had significantly lower OHP (16.5 vs. 23.1, p = 0.015) and higher OFP (72.0% vs. 65.1%, p = 0.005), as well as significantly lower AST, ferritin, LDH, CRP, and D-dimer. A multivariate model containing OHP was constructed with the outcome of oxygen requirement, with c-statistic of 0.85 (95%CI 0.81-0.90). In this pilot study, we show a significant correlation between OHP results and requirement for oxygen supplementation in hospitalized patients during a period dominated by the Omicron variant. The results were incorporated into a multivariate model that predicted for oxygen requirement, with high discriminative ability.

Thrombosis is one of the major global causes of morbidity and mortality, and predicting the risk of thrombotic and cardiovascular complications remains one of the key challenges in modern medicine. Conventional coagulation testing does not provide sufficient information, primarily because they measure the time to start of blood clotting and do not evaluate total thrombin generation. Possible adjunctive tools that may be helpful are global coagulation assays, which includes the assessment of the final products of the coagulation cascade, namely thrombin and fibrin. Whilst these assays have been more widely investigated in bleeding states, their role in thrombotic disorders is less established. We have previously investigated the use of assays such as thromboelastography, calibrated automated thrombogram and overall haemostatic potential assay in several hypercoagulable states including cardiovascular disease, haematological disorders and influence of hormone status as well as healthy controls. We provide a review of the use and limitations of global coagulation assays in healthy controls as well as hypercoagulable conditions.

Venous thromboembolism (VTE) is a leading cause of morbidity and mortality and is associated with high recurrence rates. The introduction of direct oral anticoagulants (DOACs) in the 2010s has changed the landscape of VTE management. DOACs have become the preferred anticoagulant therapy for their ease of use, predictable pharmacokinetics, and improved safety profile. Increasingly, guidelines have recommended long term anticoagulation for some indications such as following first unprovoked major VTE, although an objective individualised risk assessment for VTE recurrence remains elusive. The balance of preventing VTE recurrence needs to be weighed against the not insignificant bleeding risk, which is cumulative with prolonged use. Hence, there is a need for an individualised, targeted approach for assessing the risk of VTE recurrence, especially in those patients in whom the balance between benefit and risk of long-term anticoagulation is not clear. Clinical factors alone do not provide the level of discrimination required on an individual level. Laboratory data from global coagulation assays and biomarkers may provide enhanced risk assessment ability and are an active area of research. A review of the prediction models and biomarkers for assessing VTE recurrence risk is provided, with an emphasis on contemporary developments in the era of DOACs and global coagulation assays.

CONTEXT: As the number of transgender (trans) people (including those who are binary and/or nonbinary identified) seeking gender-affirming hormone therapy rises, endocrinologists are increasingly asked to assist with interpretation of laboratory tests. Many common laboratory tests such as hemoglobin, iron studies, cardiac troponin, and creatinine are affected by sex steroids or body size. We seek to provide a summary of the impact of feminizing and masculinizing hormone therapy on common laboratory tests and an approach to interpretation. CASES: Case scenarios discussed include 1) hemoglobin and hematocrit in a nonbinary person undergoing masculinizing hormone therapy; 2) estimation of glomerular filtration rate in a trans woman at risk of contrast-induced nephropathy; 3) prostate-specific antigen (PSA) in a trans woman; and 4) chest pain in a trans man with a cardiac troponin concentration between the reported male and female reference ranges. CONCLUSIONS: The influence of exogenous gender-affirming hormone therapy on fat and muscle distribution and other physiological changes determines interpretation of laboratory tests that have sex-specific differences. In addition to affirmative practice to ensure a patient's name, gender, and pronoun are used appropriately, we propose that once individuals have commenced gender-affirming hormone therapy, the reference range of the affirmed gender be reported (and specified by treating clinicians) except for PSA or cardiac troponin, which are dependent on organ size. While suggestions may be challenging to implement, they also represent an opportunity to lead best practice to improve the quality of care and experiences of healthcare for all trans people.

BACKGROUND: There is significant heterogeneity in the incidence and severity of diabetes-associated vascular complications and there is no routine biomarker that accurately predicts these outcomes. This pilot study investigates the role of global coagulation assays in patients with diabetes mellitus. METHODS: In this cross-sectional study, patients with diabetes not on anticoagulation or dialysis and without active malignancy were recruited from endocrinology clinics. Blood samples were collected for global coagulation assays including thromboelastography (TEG), thrombin generation using calibrated automated thrombogram (CAT), and fibrin generation and fibrinolysis using the overall hemostatic potential (OHP) assay. The results were compared with healthy controls. RESULTS: A total of 147 adult patients including 19 with type 1 diabetes (T1DM), 120 with type 2 diabetes (T2DM), and eight with latent autoimmune diabetes were recruited. Compared with 153 healthy controls, patients with diabetes demonstrated higher maximum amplitude (68.6 vs 60.2 mm, p < 0.001) on TEG, and higher OHP (9.3 vs 6.4, p < 0.001) with comparable CAT parameters. Patients with T2DM were more hypercoagulable than those with T1DM on most biomarkers. Higher maximum amplitude, velocity index, and OHP were associated with increased risk of complications (C-stat 0.82). Patients with history of microvascular complications appear to have more hypercoagulable thrombin and fibrin generation than those without. CONCLUSION: Patients with diabetes have more hypercoagulable profiles on global coagulation assays, particularly patients with T2DM and those with microvascular complications. Further studies with longitudinal follow-up are ongoing to evaluate the utility of global coagulation assays in predicting long-term patient outcomes.

INTRODUCTION: Women are at higher risk of venous thromboembolism (VTE) during pregnancy and the puerperium. Global coagulation assays (GCAs), including thromboelastography (TEG), thrombin generation using the calibrated automated thrombogram (CAT) and fibrin generation using the overall haemostatic potential assay (OHP), provide a more comprehensive assessment of the coagulation process than conventional coagulation assays. We aimed to evaluate the ability of these GCAs to analyse the coagulability among pregnant women of varying VTE risk profile. METHODS: Women undergoing term elective caesarean delivery provided a single predelivery blood sample for conventional and novel coagulation testing (TEG, CAT and OHP). Data from 47 healthy nonpregnant women aged 18-45 years were used as controls. RESULTS: Sixty women with term singleton pregnancies were included. Samples from pregnant women were hypercoagulable on most GCA parameters compared to nonpregnant controls, demonstrating increased maximum amplitude (clot strength) (71.5 vs 60.6 mm, P < .001) on whole blood TEG and increased endogenous thrombin potential (1895.22 vs 1399.33 nmol/L·min, P < .001) and overall coagulation potential (fibrin generation) (57.58 vs 36.21 units, P < .001) on platelet-poor plasma. Pregnant women with booking BMI ≥ 30 kg/m2 had significantly higher maximum amplitude compared to pregnant women of normal BMI (18.5-25 kg/m2 ) (73.2 vs 66.1 mm, P < .001). CONCLUSIONS: Global coagulation assays reliably detect the physiological hypercoagulability of pregnancy. Thromboelastography in particular appears to correlate with obesity in the pregnant population. GCAs may be potential adjuncts to risk factor-based criteria to guide VTE thromboprophylaxis during pregnancy and the puerperium.

BACKGROUND: Clinical trials have demonstrated that direct oral anticoagulants (DOAC) are non-inferior to vitamin K antagonist for stroke prevention in non-valvular atrial fibrillation (AF) with comparable safety outcomes; however, real-world Australian data are limited. AIMS: To evaluate local real-world DOAC use focussing on safety, particularly in high-risk patients. METHODS: A retrospective evaluation of 658 patients commenced or continued on DOAC between September 2013 and September 2016 for non-valvular AF at Northern Hospital, a tertiary hospital in Victoria, Australia was performed. RESULTS: Factor Xa inhibitors were more commonly prescribed than direct thrombin inhibitors (83.3 vs 16.7%) for AF management. The median patient age was 75 years. The rate of clinically significant bleeding on anticoagulation was 3.13 per 100 person-years (including four deaths) with risk factors including history of bleeding (hazard ratio (HR) 3.52, 95% confidence interval (CI) 1.22-10.17), concurrent antiplatelet therapy (HR 2.62, 95% CI: 1.11-6.20) and high falls risk (HR 2.76, 95% CI: 1.26-6.08). Patients on low-dose DOAC had significantly higher bleeding risk compared with those on full dose (5.05 vs 1.82 per 100 person-years). The rate of thrombotic stroke despite anticoagulation was 1.34 per 100 person-years with risk factors including low dose anticoagulation (P = 0.034), high falls risk (P = 0.046) and previous stroke (P = 0.028). CONCLUSIONS: DOAC use in real-world Australian practice is safe and effective, consistent with international data. Low dose anticoagulation and falls risk are associated with increased bleeding and thrombotic risk demonstrating overlapping risk factors. Careful individualised patient risk assessment is still required as low dose anticoagulation is not without risks.

INTRODUCTION: Some patients with thrombocytopenia may be at risk of bleeding although quantitative platelet count is not always a sufficient predictive factor. Global coagulation assays such as thromboelastography (TEG® ), calibrated automated thrombogram (CAT) and overall haemostatic potential (OHP) may provide a better assessment of an individual's haemostatic profile. METHODS: Blood samples were collected from thrombocytopenic patients. TEG® was performed on citrated whole blood, while CAT and OHP were performed on platelet-poor plasma. Results were compared to our previously collected normal controls. RESULTS: Fifty-eight participants (24 immune thrombocytopenia, 34 chemotherapy/malignancy-related) with mean age of 57.5 years were recruited. Compared to normal controls, thrombocytopenic participants had comparable maximum amplitude but reduced clot lysis (0.0% vs 0.6%; P < 0.001) on TEG® with reduced endogenous thrombin potential on CAT (1252.2 vs 1353.0 nmol/L/min; P = 0.040). No differences were seen in the OHP parameters. TEG® showed significant difference between marked and mild thrombocytopenia groups with minimal differences seen on CAT and OHP. Those with marked thrombocytopenia showed reduced maximum amplitude (47.2 vs 57.8 mm; P = 0.002) as expected while participants with mild thrombocytopenia (platelet count 100-150 × 109 /L) paradoxically demonstrated increased maximum amplitude (66.4 vs 57.8 mm; P < 0.001). CONCLUSION: Global coagulation assays, particularly TEG® , can detect subtle differences in coagulation in thrombocytopenic patients. While patients with marked thrombocytopenia showed reduced maximum amplitude, patients with mild thrombocytopenia appear to paradoxically show increased maximum amplitude, suggesting compensatory activity within the coagulation pathway which may in part explain why not all thrombocytopenic patients have bleeding complications.

BACKGROUND: The international, phase 3 COMPARZ study demonstrated that pazopanib and sunitinib have comparable efficacy as first-line therapy in patients with advanced renal cell carcinoma, but that safety and quality-of-life profiles favor pazopanib. Our report analyzed pazopanib and sunitinib safety in Asian and non-Asian subpopulations. METHODS: Patients were randomized 1:1 to receive pazopanib 800 mg once daily (continuous dosing) or sunitinib 50 mg once daily in 6-week cycles (4 weeks on, 2 weeks off). RESULTS: Safety population was composed of 363 Asian patients and 703 non-Asian patients. Asian patients had similar duration of exposure to either drug compared with non-Asian patients, although Asian patients had a higher frequency of dose modifications. Overall, hematologic toxicities, cytopenias, increased AST/ALT, and palmar-plantar erythrodysesthesia (PPE) were more prevalent in Asian patients, whereas gastrointestinal toxicities were more prevalent in non-Asian patients. Among Asian patients, hematologic adverse events and most non-hematologic AEs were more common in sunitinib-treated versus pazopanib-treated patients. Among Asian patients, the most common grade 3/4 AEs with pazopanib were hypertension (grade 3, 22%) and alanine aminotransferase increased (grade 3, 12%; grade 4, 1%); the most common grade 3/4 AEs with sunitinib were thrombocytopenia/platelet count decreased (grade 3, 36%; grade 4, 10%), neutropenia/neutrophil count decreased (grade 3, 24%; grade 4, 3%) hypertension (grade 3, 20%), and PPE (grade 3, 15%). CONCLUSIONS: A distinct pattern and severity of adverse events was observed in Asians when compared with non-Asians with both pazopanib and sunitinib. However, the two drugs were well tolerated in both subpopulations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00720941 , Registered July 22, 2008 ClinicalTrials.gov, NCT01147822 , Registered June 22, 2010.

BACKGROUND: Venous thromboembolism (VTE) is a major cause of morbidity and mortality with significant heterogeneity in its management, both within our local practice and in international guidelines. AIMS: To provide a holistic evaluation of 'real-world' Australian experience in the warfarin era, including how we compare to international guidelines. METHODS: Retrospective evaluation of VTE from July 2011 to December 2012 at two major hospitals in Melbourne, Australia. These results were compared to recommendations in the international guidelines. RESULTS: A total of 752 episodes involving 742 patients was identified. Contrary to international guidelines, an unwarranted heritable thrombophilia screen was performed in 22.0% of patients, amounting to a cost of AU$29 000. The duration of anticoagulation was longer compared to international recommendations, although the overall recurrence (3.2/100 person-years) and clinically significant bleeding rates (2.4/100 person-years) were comparable to 'real-world' data. Unprovoked VTE (hazard ratio 2.06; P = 0.01) was a risk factor for recurrence, and there was no difference in recurrence between major VTE (proximal deep vein thrombosis (DVT) and/or pulmonary embolism) and isolated distal DVT (3.02 vs 3.94/100 person-years; P = 0.25). Fourteen patients were subsequently diagnosed with malignancy, and patients with recurrent VTE had increased risk of prospective cancer diagnosis (relative risk 6.68; P < 0.001). CONCLUSIONS: While our 'real-world' VTE experience during the warfarin era largely correlates with international guidelines, there remains heterogeneity in the management strategies, including excessive thrombophilia screening and longer duration of anticoagulation. This audit highlights the need for national VTE guidelines, as well as prospective auditing of VTE management, in the direct oral anticoagulant era for future comparison.

OBJECTIVES: Venous thromboembolism (VTE) provoked by transient risk factors has traditionally been classified as a single entity with lower risk of recurrence. We evaluated the association between different categories of transient provoking factors and the relative risk of recurrence. METHODS: Retrospective evaluation of VTE events in non-cancer patients from July 2011 to December 2012 at two tertiary institutions in Australia with a minimum follow-up of 24 months. RESULTS: A total of 747 VTE cases were identified, and following exclusion of cases with mortality within 30 days of presentation (n=26), unprovoked cases (40.2%) had a higher risk of recurrence (4.6 vs 2.3/100 event-years, P=.01). Provoking factors included surgery (40.4%), injury (16.7%), medical-related factors including non-surgical hospitalisation or active infection (22.0%), travel (13.2%) and oestrogen related (6.5%). Air travel had the highest recurrence rate of 5.9/100 event-years, comparable to unprovoked VTE. VTE provoked by surgery showed lower recurrence rate at 1.8/100 event-years (P=.03). 62.5% of patients with provoked VTE recurred with an unprovoked event. CONCLUSION: Transient provoking factors for VTE are heterogeneous with varying potency and should not be considered a single entity. The high recurrence rate after travel-provoked VTE suggests that it is a "minor," if not negligible provoking factor with higher thrombotic predisposition.

Direct oral anticoagulants (DOACs) have led to a paradigm shift in the field of anticoagulation, providing safe and convenient anticoagulation without the need for regular blood testing. Currently, there are three major DOACs available-Factor Xa inhibitors (apixaban and rivaroxaban) and direct thrombin inhibitors (dabigatran)-that are available for use in atrial fibrillation and venous thromboembolism. While these agents have been shown to be as effective as warfarin, with a similar or better bleeding profile, there remains some concern of the use of these drugs in vulnerable populations, such as the frail elderly patients; particularly since reversal agents and drug monitoring are not routinely available. We aim to provide a review of the use of DOACs and the impact of DOACs on dental treatment in the elderly population.

Removal of cholesterol from peripheral tissues to the bloodstream via reverse cholesterol transport (RCT) is a process of major biological importance. Here we demonstrate that lymphatic drainage is required for RCT. We have previously shown that hypercholesterolemia in mice is associated with impaired lymphatic drainage and increased lipid accumulation in peripheral tissues. We now show that restoration of lymphatic drainage in these mice significantly improves cholesterol clearance. Conversely, obstruction of lymphatic vessels in wild-type mice significantly impairs RCT. Finally, we demonstrate using silencing RNA interference, neutralizing antibody, and transgenic mice that removal of cholesterol by lymphatic vessels is dependent on the uptake and transcytosis of HDL by scavenger receptor class B type I expressed on lymphatic endothelium. Collectively, this study challenges the current view that lymphatic endothelium is a passive exchange barrier for cholesterol transport and provides further evidence for its interplay with lipid biology in health and disease. © 2013 Elsevier Inc.

The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35(nck5alpha) or its isoform, p39(nck5ai). To identify novel p35(nck5alpha) - and p39(nck5alphai)-binding proteins, fragments of p35(nck5alpha) and p39(nck5alphai) were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35(nck5alpha) and p39(nck5alphai). Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35(nck5alpha) through its binding to p35(nck5alpha). The p35(nck5alpha)-interacting region was mapped to a predicted a-helix in SET. When cotransfected into COS-7 cells, SET and p35(nck5alpha) displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/p35(nck5alpha) from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35(nck5alpha), but not that of Cdk5/p25(nck5alpha) was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p25(nck5alpha).

Although Cdk5 shows high sequence identity to Cdk1 and Cdk2, it can be fully activated by its neuronal activators p35/p25(nck5a) and p39(nck5ai) in a phosphorylation-independent manner. To understand structural basis of the Cdk5/p25(nck5a) activation, the complex is modelled to assume either an obstructed or an opened conformation based on X-ray structures of the unphosphorylated or the phosphorylated Cdk2/cyclin A complex, respectively. Comparison and analysis of the two models, along with mutagenesis studies of p25(nck5a),suggest that the opened form represents more closely the structure of active Cdk5/p25(nck5a), The results provide a rationale basis for understanding the phosphorylation-independent activation of Cdk5/p25(nck5a) (C) 2001 Academie Press.