NETs and Deep vein thrombosis

Deep vein thrombosis (DVT) is a major health problem that requires improved prophylaxis and treatment.Inflammatory conditions such as infection, cancer, and autoimmune diseases are risk factors for DVT. We and othershave recently shown that extracellular DNA fibers produced in inflammation and known as neutrophil extracellulartraps (NETs) contribute to experimental DVT. NETs stimulate thrombus formation and coagulation and are abundant inthrombi in animal models of DVT. It appears that, in addition to fibrin and von Willebrand factor, NETs represent a third

thrombus scaffold. Here, we review how NETs stimulate thrombosis and discuss known and potential interactions ofNETs with endothelium, platelets, red blood cells, and coagulation factors and how NETs could influence thrombolysis.It was proposed that drugs that inhibit NET formation or facilitate NET degradation may prevent or treat DVT. 

Deep vein thrombosis (DVT) is a debilitating disease that may be complicated by pulmonary embolism (PE). Together DVT and PE are designated as venous thromboembolism. In the United States, venous thromboembolism develops in an estimated 900000 patients each year, and PE is responsible for ≈300000 deaths, which exceeds the mortality from myocardial infarction or stroke.DVT complications, in addition to PE, include post thrombotic syndrome caused by chronic venous stasis even in the absence of active thrombosis

NETs are produced to allow neutrophils to trap and disarm microbes in the extracellular environment. NETs are scaffolds of intact chromatin fibers with antimicrobial proteins, ideal to retain large quantities of microbes. Therefore, some pathogenic bacteria have evolved to express an extracellular deoxyribonuclease (DNase), which dismantles NETs and promotes virulence. Extracellular traps are formed in humans, animals, and even plants, indicating that NETs provide an evolutionary conserved protective mechanism. 

NETs formation is not restricted to neutrophils, and different cell types use different cellular mechanisms to release extracellular trap. One mechanism used by human neutrophils is NETosis. NETosis is a multistep cell death program . On activation, certain enzymes translocate from the granules to the nucleus. Histones are degraded by neutrophil elastase (NE) and citrullinated by peptidylarginine deiminase 4 to unwind chromatin. Further hallmarks are the breakdown of granular and nuclear membranes and cytolysis as the final step in NETosis.

Implications of NETs in Thrombolysis -To degrade and solubilize thrombi to restore blood flow, fibrin  and VWF as the main scaffolds need to be proteolytically fragmented by the proteases plasmin and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13,  respectively. NETs are newly recognized third scaffolds that need to be undone during thrombolysis (Figure 1C). NETs were seen to colocalize with fibrin in clots 15 and with VWF in venous thrombi. In vitro, we could show that NETs provide a scaffold for blood clots that is resistant to tPA-induced thrombolysis. We incubated recalcified blood with neutrophils which were prestimulated to release NETs. As shown in  Figure 3, after filtration, blood clots appeared in control samples and tPA- or DNase-treated blood but not in blood treated with the combination of tPA and DNase. Immunostainings revealed that in the presence of tPA, blood clots lacked fibrin and were held together by a scaffold  of extracellular DNA (Figure 3B). DNase1 is the predominant nuclease in plasma. Interestingly, the plasminogen system cooperates with DNase1 during chromatin degradation. DNase1 has only limited activity to degrade chromatin because it preferentially degrades protein-free DNA. Plasminogen, activated by either tPA or urokinase-type plasminogen activator, degrades histones and therefore allows for degradation of DNA by DNase1. Monocytes/macrophages may also support the DNA degradation because their lysosomes contain DNase2, which is important for the removal of apoptotic cells (Figure 1C). NETs and fibrin degradation by plasmin and DNase could result in the simultaneous release of DNA and fibrin fragments.  In baboon DVT, plasma DNA increases with similar kinetics to the fibrin degradation product D-dimers. Recently, in collaboration with Thomas Wakefield’s group, we found increased levels of DNA in plasma from patients with DVT compared with healthy controls and symptomatic patients who did not have DVT. Here also, plasma DNA con-centrations correlated with D-dimers (unpublished data; Diaz and Fuchs, 2012). Therefore, it is plausible that circulating DNA may reflect the degradation of NETs within a thrombus.NETs may also promote thrombolysis. In vitro studies have shown that NE and cathepsin G can degrade fibrin, and these proteases are present on NETs and could enhance fibrinolysis in DVT. In addition, NETs may also recruit plasminogen from the plasma. Histone H2B can serve as a receptor for plasminogen on the surface of human monocytes/macrophages and perhaps could do so in NETs.

hepPTFE AVGs failed to improve patency or decrease secondary interventions compared to standard PTFE grafts used as Arteriovenous grafts for dialysis

Vascular occlusions in the lower limbs require a bypass operation. A conduit is needed for the bypass operation. Autogenous vein graft from the contralateral limb or ipsilateral limb is considered as an ideal conduit for the bypass operations. But it is not available or inadequate in 20-30% of the patients requiring bypass operation. Then one has to use the synthetic vascular graft in the absence of autogenous vein graft. The synthetic vascular grafts are modified over a period of time to improve the patency and reduce the recurrent thrombosis and also reduce the need for reinterventions. Porous dacron grafts need preclotting and that is not needed in the PTFE grafts. The kinking and rotation of the synthetic grafts in the long subcutaneous tunnels is avoided  by external support (rings/spirals). In a similar way there were many attempts to make the inner surface of the graft less thrombogenic and heparin bonding (coating) was one of them. These grafts have initially shown better results in the literature and they are available in the market. But there were not many papers to establish the indications and evaluating the long term results. Now these vascular grafts are also used for creating AV fistula for  patients requiring hemodialysis and known as arterio-venous grafts. The long term patency of the A-Vgrafts without re-interventions is a boon for the patients. It was hoped that the heparin bonding to the internal luminal surface of the Arterio-Venous grafts may prevent the thrombosis.

Recently a paper is published saying that Heparin Bonding Does Not Improve Patency of Polytetrafluoroethylene Arterio-Venous Grafts by Matthew TA et al (Feb 2013). A total of 223 patients had 265 grafts placed. Of these, 62 (23%) were hepPTFE grafts. The average age was 66 ± 15 years in the hepPTFE group and 59 ± 17 years in the non–heparin-bonded control group (PTFE; P < 0.01). Of the hepPTFE group, 39% were men, 81% were African American, 63% were diabetic, and 81% had a tunneled catheter at the time of access placement. Of the PTFE group, 35% were men, 85% were African American, 56% were diabetic, and 83% had a tunneled catheter. HepPTFE grafts failed to improve rates of primary, assisted primary, or secondary patency based on univariate analysis (hazard ratio [HR]: 1.37 [95% confidence interval {CI}: 0.99–1.88]; HR: 1.39 [95% CI: 0.98–1.96]; and HR: 1.20 [95% CI: 0.73–1.96], respectively). The number of secondary interventions was similar in the 2 groups (1.1 interventions per person-year of follow-up PTFE versus 1.4 hepPTFE; P = 0.13). A multivariable model including age, diabetes, peripheral artery disease, tobacco use, previous access placement, and tunneled catheter found that the HR for hepPTFE was not significantly different than PTFE in primary, assisted primary, or secondary patency (HR: 1.32 [95% CI: 0.91–1.90]; HR: 1.35 [95% CI: 0.91–1.99]; and HR: 1.15 [95% CI: 0.62–2.16], respectively.
This probably indicates that the intraluminal thrombosis of Arterio-Venous Grafts (AVGs) in the patients undergoing Dialysis is dependent on many other factors other than less thrombogenisity of the intraluminal surface of the grafts.

A 15-fold increase in rates of mortality due to cardiovascular disease and coronary heart disease among subjects with large-vessel peripheral arterial disease !!! do you believe?

Peripheral arterial disease (PAD) is a widespread vascular disorder that has been addressed for over a century and continues to affect a large portion of the modernized world. Both symptomatic and asymptomatic PAD affects 4.3% of the U.S. population aged ≥40 years of age1 and is recognized as a chronic atherosclerotic progression of lower-extremity arterial obstruction, which eventually leads to limb-threatening ischemia. PAD is functionally defined as an occlusive disease that generates a resting ankle–brachial index (ABI) of ≤0.90,2 although an ABI of between 0.9 and 1 is considered borderline and may introduce diagnostic subjectivity. PAD is strongly associated with terminal coronary artery disease for patients both with and without a significant cardiovascular history.3 As defined by a history of cardiovascular events or interventions (abdominal aortic aneurysms, transient ischemic attacks, stroke, carotid endarterectomy, history of angina, myocardial infarction, coronary angioplasty, and/or coronary artery bypass graft surgery), general cardiovascular disease has been associated with 70% of patients with PAD, rendering its diagnosis a significant indication for pan-vascular risk.4 Thus, the timely detection of PAD permits treatment of the diseased limb and preemptive management of cardiovascular risks.5 Preliminary PAD screenings have evolved into routine, noninvasive vascular laboratory studies, which reduce the risks, time, and costs associated with angiography. 

In a 10 years followup study published in Annals of vascular surgery in 1992 it was found that  - Twenty-one of the 34 men (61.8 percent) and 11 of the 33 women (33.3 percent) with large-vessel peripheral arterial disease died during follow-up, as compared with 31 of the 183 men (16.9 percent) and 26 of the 225 women (11.6 percent) without evidence of peripheral arterial disease. After multivariate adjustment for age, sex, and other risk factors for cardiovascular disease, the relative risk of dying among subjects with large-vessel peripheral arterial disease as compared with those with no evidence of such disease was 3.1 (95 percent confidence interval, 1.9 to 4.9) for deaths from all causes, 5.9 (95 percent confidence interval, 3.0 to 11.4) for all deaths from cardiovascular disease, and 6.6 (95 percent confidence interval, 2.9 to 14.9) for deaths from coronary heart disease. The relative risk of death from causes other than cardiovascular disease was not significantly increased among the subjects with large-vessel peripheral arterial disease. After the exclusion of subjects who had a history of cardiovascular disease at base line, the relative risks among those with large-vessel peripheral arterial disease remained significantly elevated. Additional analyses revealed a 15-fold increase in rates of mortality due to cardiovascular disease and coronary heart disease among subjects with large-vessel peripheral arterial disease that was both severe and symptomatic.

1. Selvin E, Erlinger TP. Prevalence of and risk factors for peripheral arterial disease in the United States: results from the National Health and Nutrition Examination Survey, 1999–2000. Circulation. 2004;110:738–743
2. Norgren L, Hiatt WR, Dormandy JA, et al. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). J Vasc Surg. 2007;45(Suppl. S):S5–67
3. Criqui MH, Langer RD, Fronek A, et al. Mortality over a period of 10 years in patients with peripheral arterial disease. N Engl J Med. 1992;326:381–386
4. Hirsch AT, Criqui MH, Treat-Jacobson D, et al. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA. 2001;286:1317–1324
5. Verhaeghe R. Prophylactic antiplatelet therapy in peripheral arterial disease. Drugs. 1991;42((Suppl. 5)):51–57

We should check the ABI in all patients at risk of peripheral arterial disease!

International ABI awareness as the next step in the PAD campaign

Coronary artery disease, cerebrovascular disease are well known in the society as the cause for heart attack (MI) and brain attack (stroke). Peripheral artery disease is the third most common manifestation of the atherosclerosis and one can lose lower limb if the critical ischemia is precipitated by other factors. The awareness of peripheral vascular disease is not adequate enough among the people in our society or general practioners to avoid complications and toe or limb loss in India and many other countries.

Peripheral artery disease (PAD) is common, underdiagnosed, and undertreated. Owing to the systemic nature of atherosclerosis, PAD patients are at risk for polyvascular disease. For example, 63% of patients with PAD have concomitant symptomatic cerebrovascular or coronary disease. Accordingly, PAD patients are at significantly increased risk for myocardial infarction, stroke, and vascular death over a 5-year period compared to age-matched cohorts.  

The ankle–brachial index (ABI) is the preferred initial test for PAD screening and diagnosis. It is relatively inexpensive, sensitive, and specific. Current guidelines provide clear recommendations on the indications for ABI testing. However, these guidelines may not have been fully implemented among practitioners.

In our practice we rarely see patients getting referred based on the ABI recorded in the clinics. The clinicians ask for Colour Doppler study (both legs costing Rs 2000 to 3000) and then send them with a report saying diffuse peripheral vascular disease in the diabetic and smoking population. Then we are doing the ankle brachial index in our clinic to classify degree of ischemia. One should practice checking the ankle brachial index routinely in patients with suspected peripheral arterial disease.

In a survey conducted in Australia, it was found that strikingly low 6% of GPs were aware of evidence-based guidelines on PAD screening, and only 5% were aware of guidelines on PAD diagnosis. The majority of GPs (58%) never perform ABIs. Most notably, 70% of the respondents choose arterial duplex (which is more costly and time-consuming) as the initial diagnostic tool in a patient with a history and physical exam consistent with PAD; younger GPs were more likely to choose the ABI. I think we are no better than the GPs in Australia in the evaluation of Peripheral vascular disease in the community.

The most common ‘moderate to major’ barriers to PAD screening and testing were (1) equipment availability, (2) time constraints, (3) lack of training and skills, and (4) staff availability. The time constraint barrier is not surprising, given that the time for an ABI could approach the 15-minute length of a typical primary care office visit. Other studies have also identified limited reimbursement and time as primary barriers to widespread use of the ABI in primary care practices.

I think, by increasing the awareness and improving staff ability more and more GPs will make an attempt to record the ABI in their practice and follow their patients for the CV events and extend better protection measures to avoid the amputations.