Development of endothelial-targeted CD39 as a therapy for ischemic stroke.

BACKGROUND: Ischemic stroke is characterized by a necrotic lesion in the brain surrounded by an area of dying cells termed the penumbra. Salvaging the penumbra either with thrombolysis or mechanical retrieval is the cornerstone of stroke management. At-risk neuronal cells release extracellular adenosine triphosphate, triggering microglial activation and causing a thromboinflammatory response, culminating in endothelial activation and vascular disruption. This is further aggravated by ischemia-reperfusion injury that follows all reperfusion therapies. The ecto-enzyme CD39 regulates extracellular adenosine triphosphate by hydrolyzing it to adenosine, which has antithrombotic and anti-inflammatory properties and reverses ischemia-reperfusion injury. OBJECTIVES: The objective off the study was to determine the efficacy of our therapeutic, anti-VCAM-CD39 in ischaemic stroke. METHODS: We developed anti-VCAM-CD39 that targets the antithrombotic and anti-inflammatory properties of recombinant CD39 to the activated endothelium of the penumbra by binding to vascular cell adhesion molecule (VCAM)-1. Mice were subjected to 30 minutes of middle cerebral artery occlusion and analyzed at 24 hours. Anti-VCAM-CD39 or control agents (saline, nontargeted CD39, or anti-VCAM-inactive CD39) were given at 3 hours after middle cerebral artery occlusion. RESULTS: Anti-VCAM-CD39 treatment reduced neurologic deficit; magnetic resonance imaging confirmed significantly smaller infarcts together with an increase in cerebrovascular perfusion. Anti-VCAM-CD39 also restored blood-brain barrier integrity and reduced microglial activation. Coadministration of anti-VCAM-CD39 with thrombolytics (tissue plasminogen activator [tPA]) further reduced infarct volumes and attenuated blood-brain barrier permeability with no associated increase in intracranial hemorrhage. CONCLUSION: Anti-VCAM-CD39, uniquely targeted to endothelial cells, could be a new stroke therapy even when administered 3 hours postischemia and may further synergize with thrombolytic therapy to improve stroke outcomes.

Overall haemostatic potential assay for prediction of outcomes in venous and arterial thrombosis and thrombo-inflammatory diseases.

Thromboembolic diseases including arterial and venous thrombosis are common causes of morbidity and mortality globally. Thrombosis frequently recurs and can also complicate many inflammatory conditions through the process of 'thrombo-inflammation,' as evidenced during the COVID-19 pandemic. Current candidate biomarkers for thrombosis prediction, such as D-dimer, have poor predictive efficacy. This limits our capacity to tailor anticoagulation duration individually and may expose lower risk individuals to undue bleeding risk. Global coagulation assays, such as the Overall Haemostatic Potential (OHP) assay, that investigate fibrin generation and fibrinolysis, may provide a more accurate and functional assessment of hypercoagulability. We present a review of fibrin's critical role as a central modulator of thrombotic risk. The results of our studies demonstrating the OHP assay as a predictive biomarker in venous thromboembolism, chronic renal disease, diabetes mellitus, post-thrombotic syndrome, and COVID-19 are discussed. As a comprehensive and global measurement of fibrin generation and fibrinolytic capacity, the OHP assay may be a valuable addition to future multi-modal predictive tools in thrombosis.

Immune cell expression patterns of CD39/CD73 ectonucleotidases in rodent models of cardiac arrest and resuscitation.

Background: Cardiac arrest (CA) is a significant public health concern. There is the high imminent mortality and survival in those who are resuscitated is substantively compromised by the post-CA syndrome (PCAS), characterized by multiorgan ischemia-reperfusion injury (IRI). The inflammatory response in PCAS is complex and involves various immune cell types, including lymphocytes and myeloid cells that have been shown to exacerbate organ IRI, such as myocardial infarction. Purinergic signaling, as regulated by CD39 and CD73, has emerged as centrally important in the context of organ-specific IRI. Hence, comprehensive understanding of such purinergic responses may be likewise imperative for improving outcomes in PCAS. Methods: We have investigated alterations of immune cell populations after CA by utilizing rodent models of PCAS. Blood and spleen were collected after CA and resuscitation and underwent flow cytometry analysis to evaluate shifts in CD3+CD4+ helper T cells, CD3+CD8a+ cytotoxic T cells, and CD4/CD8a ratios. We then examined the expression of CD39 and CD73 across diverse cell types, including myeloid cells, T lymphocytes, and B lymphocytes. Results: In both rat and mouse models, there were significant increases in the frequency of CD3+CD4+ T lymphocytes in PCAS (rat, P < 0.01; mouse, P < 0.001), with consequently elevated CD4/CD8a ratios in whole blood (both, P < 0.001). Moreover, CD39 and CD73 expression on blood leukocytes were markedly increased (rat, P < 0.05; mouse, P < 0.01 at 24h). Further analysis in the experimental mouse model revealed that CD11b+ myeloid cells, with significant increase in their population (P < 0.01), had high level of CD39 (88.80 ± 2.05 %) and increased expression of CD73 (P < 0.05). CD19+ B lymphocytes showed slight increases of CD39 (P < 0.05 at 2h) and CD73 (P < 0.05 at 2h), while, CD3+ T lymphocytes had decreased levels of them. These findings suggested a distinct patterns of expression of CD39 and CD73 in these specific immune cell populations after CA. Conclusions: These data have provided comprehensive insights into the immune response after CA, highlighting high-level expressions of CD39 and CD73 in myeloid cells.

Association Between Thromboelastometry Identified Hypercoagulability and Thromboembolic Complications After Arthroplasty: A Prospective Observational Study in Patients With Obesity.

The prothrombotic state of obesity can increase the risk of thromboembolism. We aimed to investigate if there was an association between baseline hypercoagulable rotational thromboelastometry (ROTEM) profile and thromboembolic complications in arthroplasty patients with obesity. Patients with a body mass index ≥ 25 kg/m2 and/or waist circumference ≥94 cm (M) and 80 cm (F) undergoing hip and knee arthroplasty had pre- and postoperative ROTEM. ROTEM values were compared by outcome status using an independent sample equal-variance t-test. Of the 303 total participants, hypercoagulability defined as extrinsically activated thromboelastometry maximum clot firmness G score ≥ 11 K dyne/cm2, was observed in 90 (30%) of the 300 participants with preoperative ROTEM assays. Clinically significant thromboembolic complications occurred in 5 (1.7%) study participants before discharge and in 10 (3.3%) by 90 days. These included 6 with pulmonary emboli, 3 with deep venous thrombus, and 1 with myocardial infarction. We found no evidence for an association between baseline hypercoagulability and incident thromboembolic events, analysis limited by the number of events. Postoperative decrease in platelets and an increase in fibrinogen were observed. ROTEM parameter changes differed across obesity categories.

Current challenges in the prevention and management of post-thrombotic syndrome-towards improved prevention.

Post-thrombotic syndrome (PTS) is a common and potentially debilitating complication of deep vein thrombosis (DVT), affecting up to 50% of DVT patients. The consequence of this chronic condition includes reduced quality of life, increased use of the healthcare system and decreased productivity. The societal impact of this condition is projected to increase, given our ageing population and increased burden of thrombotic diseases. Despite significant recent advances in our understanding of PTS, many unanswered questions remain. Currently, there are few effective and proven options for established PTS; hence, the emphasis should be on instituting effective prevention to reduce the progression to PTS. Effective anticoagulation lowers the risk of PTS, with direct oral anticoagulants appearing to outperform vitamin-K antagonists. However, the evidence for elastic compression stockings and endovascular thrombolysis or thrombectomy techniques remains unclear. Accurate identification of individuals at high risk of developing PTS may also improve the targeting of preventative interventions. This review will examine the current body of evidence regarding PTS, with a focus on preventative strategies as well as novel biomarkers.

Rotational ThromboElastometry-guided blood component administration versus standard of care in patients with Cirrhosis and coagulopathy undergoing Invasive ProcEdures (RECIPE): study protocol for a randomised controlled trial.

BACKGROUND: Patients with cirrhosis often undergo invasive procedures both for management of complications of their advanced liver disease, including treatment for hepatocellular carcinoma, as well as underlying comorbidities. Despite a current understanding that most patients with cirrhosis are in a rebalanced haemostatic state (despite abnormalities in conventional coagulation tests, namely INR and platelet count), patients with cirrhosis are still often given prophylactic blood components based on these conventional parameters, in an effort to reduce procedure-related bleeding. Viscoelastic tests such as Rotational Thromboelastometry (ROTEM) provide a global measurement of haemostasis and have been shown to predict bleeding risk more accurately than conventional coagulation tests, and better guide blood product transfusion in a number of surgical and trauma-related settings. The aim of this study is to assess the utility of a ROTEM-based algorithm to guide prophylactic blood component delivery in patients with cirrhosis undergoing invasive procedures. We hypothesise that ROTEM-based decision-making will lead to a reduction in pre-procedural blood component usage, particularly fresh frozen plasma (FFP), compared with standard of care, whilst maintaining optimal clinical outcomes. METHODS: This is a multi-centre randomised controlled trial comparing ROTEM-guided prophylactic blood component administration to standard of care in patients with cirrhosis and coagulopathy undergoing invasive procedures. The primary efficacy outcome of the trial is the proportion of procedures requiring prophylactic transfusion, with the primary safety outcome being procedure-related bleeding complications. Secondary outcomes include the amount of blood products (FFP, platelets, cryoprecipitate) transfused, transfusion-related side effects, procedure-related complications other than bleeding, hospital length of stay and survival. DISCUSSION: We anticipate that this project will lead to improved prognostication of patients with cirrhosis, in terms of their peri-procedural bleeding risk. We hope to show that a significant proportion of cirrhotic patients, deemed coagulopathic on the basis of standard coagulation tests such as INR and platelet count, are actually in a haemostatic balance and thus do not require prophylactic blood product, leading to decreased and more efficient blood component use. TRIAL REGISTRATION: RECIPE has been prospectively registered with the Australia and New Zealand Clinical Trials Registry on the 30th April 2019 ( ACTRN12619000644167 ).

The role of pyruvate-induced enhancement of oxygen metabolism in extracellular purinergic signaling in the post-cardiac arrest rat model.

Purine nucleotide adenosine triphosphate (ATP) is a source of intracellular energy maintained by mitochondrial oxidative phosphorylation. However, when released from ischemic cells into the extracellular space, they act as death-signaling molecules (eATP). Despite there being potential benefit in using pyruvate to enhance mitochondria by inducing a highly oxidative metabolic state, its association with eATP levels is still poorly understood. Therefore, while we hypothesized that pyruvate could beneficially increase intracellular ATP with the enhancement of mitochondrial function after cardiac arrest (CA), our main focus was whether a proportion of the raised intracellular ATP would detrimentally leak out into the extracellular space. As indicated by the increased levels in systemic oxygen consumption, intravenous administrations of bolus (500 mg/kg) and continuous infusion (1000 mg/kg/h) of pyruvate successfully increased oxygen metabolism in post 10-min CA rats. Plasma ATP levels increased significantly from 67 ± 11 nM before CA to 227 ± 103 nM 2 h after the resuscitation; however, pyruvate administration did not affect post-CA ATP levels. Notably, pyruvate improved post-CA cardiac contraction and acidemia (low pH). We also found that pyruvate increased systemic CO2 production post-CA. These data support that pyruvate has therapeutic potential for improving CA outcomes by enhancing oxygen and energy metabolism in the brain and heart and attenuating intracellular hydrogen ion disorders, but does not exacerbate the death-signaling of eATP in the blood.

Global coagulation assays in patients with chronic kidney disease and their role in predicting thrombotic risk.

BACKGROUND: Despite cardiovascular diseases and thrombosis being major causes of death in patients with chronic kidney disease (CKD), there remains no effective biomarker to predict thrombotic risk in this population. OBJECTIVE: To evaluate global coagulation assays in patients with CKD and correlate the biomarkers to clinical outcomes. MATERIAL AND METHODS: Patients with eGFR<30 mL/min/1.73m2 were recruited (n = 90) in this prospective observational study. Blood samples were collected for global coagulation assays, including thromboelastography, calibrated automated thrombogram (CAT), overall hemostatic potential (OHP) and tissue factor pathway inhibitor (TFPI). RESULTS: Following adjustment for age and gender, CKD subjects (mean age 66 years, 36 % female) had increased maximum amplitude on thromboelastography (70.1 vs 60.2 mm, p < 0.001), higher peak thrombin (233.2 vs 219.7 mm, p = 0.030) and increased OHP (16.1 vs 6.4 units, p < 0.001) compared to healthy controls (n = 153). TFPI was also increased in CKD patients (36.4 vs 14.5 ng/mL, p < 0.001). Compared to hemodialysis patients (n = 43), peritoneal-dialysis patients (n = 25) had more hypercoagulable parameters. Thirty-five CKD patients reported thrombotic complications - key predictors included dialysis, higher fibrinogen, reduced endogenous thrombin potential, elevated D-dimer and increased TFPI. Using the dialysis cohort, the predictive risk model based on the key predictors performed better than Framingham heart score and number of cardiovascular risk factors (Harrell's C-stat 0.862 vs 0.585 vs 0.565). CONCLUSION: CKD appears to confer a hypercoagulable state compared to healthy controls. Interestingly, reduced thrombin generation and raised TFPI was paradoxically associated with increased thrombotic risks, highlighting possible complex compensatory mechanisms within the coagulation system, which may be important in predicting clinical outcomes.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

DOAC-stop can remove direct oral anticoagulants and allow analysis by global coagulation assays.

INTRODUCTION: Activated charcoal based compounds such as DOAC-stop™ (DS) have been developed to remove direct oral anticoagulant (DOAC) interference in-vitro. However, few studies have used this approach with global coagulation assays (GCAs), such as thrombin generation assays, which are sensitive to the effect of DOACs. METHODS: Thrombin generation with and without thrombomodulin (TM) via the automated ST-Genesia system, and the overall haemostatic potential (OHP) assay, a spectrophotometric fibrin generation assay 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) were measured on (i) pooled normal plasma (PNP) spiked with varying amounts of rivaroxaban, apixaban, and dabigatran, and (ii) platelet poor plasma (PPP) from 21 non-anticoagulated adults, before and after DS addition. RESULTS: Following the addition of DS to spiked PNP without thrombomodulin, thrombin and velocity index increased by 21.9% and 42.6%, respectively, while ETP increased by 6.93%. A decrease in OCP (-10.6%) and OHP (-12.7%) was observed following DS. Similar changes were seen post-DS to plasma from non-anticoagulated patients. Also in this group, pre- and post-DS thrombin generation parameters showed high correlation, with the strongest observed for ETP (R2  = 0.94). There was a strong correlation for OHP parameters, with the closest seen with OCP (R2  = 0.96) and OHP (R2  = 0.95). CONCLUSION: DS causes some changes to the ETP and OHP assay, however, strong correlations were seen pre- and post-DS in all GCA parameters. These findings support the use of DS to facilitate GCA testing in anticoagulated individuals for evaluation of the underlying thrombotic state.

Overall haemostatic potential (OHP) assay can risk stratify for venous thromboembolism recurrence in anticoagulated patients.

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.

Overall Hemostatic Potential Assay Detects Risk of Progression to Post-Thrombotic Syndrome in Anticoagulated Patients following Deep Vein Thrombosis.

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.

Obesity and the Risk of Venous Thromboembolism after Major Lower Limb Orthopaedic Surgery: A Literature Review.

The risk of venous thromboembolism following total joint arthroplasty is significantly greater than those of other types of elective orthopaedic procedures. This risk is increased in obesity due to the associated prothrombotic physiological and hematological changes that predispose to embolic events. The prevalence of obesity is increasing in the aging population, which contributes to a further increase in the risk of postoperative thrombosis in the older patients. There is a lack of clear evidence regarding dosing information for thromboprophylaxis medications in patients with obesity. As a result, the currently available thromboprophylaxis guidelines do not provide specific recommendations for this group. Suboptimal dosing regimens for these medications can place these patients at a risk of bleeding or clotting complications postsurgery. Hence any increase in dosage may require intensive surveillance for the residual anticoagulant effects and careful balancing of risks and benefits on an individual basis. Our review discusses the basis for increased thrombotic risk in obesity, the evidence supporting dosage recommendations, and the implications of the current guidelines for pharmacological thromboprophylaxis in patients with obesity undergoing lower limb arthroplasty.

Plasma Angiotensin Converting Enzyme 2 (ACE2) Activity in Healthy Controls and Patients with Cardiovascular Risk Factors and/or Disease.

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.

CD39 in the development and progression of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating progressive disease characterised by pulmonary arterial vasoconstriction and vascular remodelling. Endothelial dysfunction has emerged as a contributing factor in the development of PAH. However, despite progress in the understanding of the pathophysiology of this disease, current therapies fail to impact upon long-term outcomes which remain poor in most patients. Recent observations have suggested the disturbances in the balance between ATP and adenosine may be integral to the vascular remodelling seen in PAH. CD39 is an enzyme important in regulating these nucleos(t)ides which may also provide a novel pathway to target for future therapies. This review summarises the role of adenosine signalling in the development and progression of PAH and highlights the therapeutic potential of CD39 for treatment of PAH.

The Need for Individualized Risk Assessment in Cardiovascular Disease.

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.

The potential role of protease systems in hemophilic arthropathy.

Hemophilic arthropathy (HA) is characterized by joint damage following recurrent joint bleeds frequently observed in patients affected by the clotting disorder hemophilia. Joint bleeds or hemarthroses trigger inflammation in the synovial tissue, which promotes damage to the articular cartilage. The plasminogen activation system is integral to fibrinolysis, and the urokinase plasminogen activator, or uPA in particular, is strongly upregulated following hemarthroses. uPA is a serine protease that catalyzes the production of plasmin, a broad-spectrum protease that can degrade fibrin as well as proteins of the joint extracellular matrix and cartilage. Both uPA and plasmin are able to proteolytically generate active forms of matrix metalloproteinases (MMPs). The MMPs are a family of >20 proteases that are secreted as inactive proenzymes and are activated extracellularly. MMPs are involved in the degradation of all types of collagen and proteoglycans that constitute the extracellular matrix, which provides structural support to articular cartilage. The MMPs have an established role in joint destruction following rheumatoid arthritis (RA). They degrade cartilage and bone, indirectly promoting angiogenesis. MMPs are also implicated in the pathology of osteoarthritis (OA), characterized by degradation of the cartilage matrix that precipitates joint damage and deformity. HA shares a number of overlapping pathological characteristics with RA and OA. Here we discuss how the plasminogen activation system and MMPs might exacerbate joint damage in HA, lending insight into novel possible therapeutic targets to reduce the comorbidity of hemophilia.

Age, sex and racial differences in fibrin formation and fibrinolysis within the healthy population.

Increased fibrin generation and reduced fibrinolytic potential have been detected using global coagulation assays in several hypercoagulable states including cardiovascular disease and venous thromboembolism. We aimed in this study to define the impact of age, sex and race on fibrin generation and lysis using the Overall Haemostatic Potential (OHP) assay in a group of stringently defined healthy adults. Healthy adult patients not receiving anticoagulation and without a history of thrombotic disease were prospectively recruited. Iindividuals with cardiovascular risk factors (e.g. hypertension, diabetes, smoking), receiving hormonal therapy, antiplatelet agents or with abnormal routine blood tests were also excluded. Platelet-poor plasma was obtained and the OHP assay, which evaluates fibrin formation with and without tissue plasminogen activator, was performed on all plasma samples. 144 healthy subjects (34.7% male) with median age 42 years (interquartile range 20, 77) were recruited. After multivariate analysis, age at least 50 years and female sex were associated with significantly increased fibrin generation parameters (overall coagulation potential, OHP, maximum optical density, fibrin) as well as reduced markers of fibrinolysis (overall fibrinolytic potential and time-to-50% lysis). There were no significant differences in OHP parameters between whites, East Asians and South Asians after accounting for age and sex. This study defines age, sex and racial differences of fibrin generation and fibrinolysis as measured by the OHP assay in a sample of healthy subjects. Further studies are warranted in diseased populations, where there is growing awareness of the role of global coagulation assay in defining prothrombotic and hypofibrinolytic states.