The care of transmetatarsal amputation in diabetic foot gangrene.

Diabetic foot ulcerations may determine minor or major amputation, with a high impact on patients' life expectation and quality of life and on economic burden. Among minor amputations, transmetatarsal amputation (TMA) appears to be the most effective in terms of limb salvage rates and in maintaining foot and ankle biomechanics. In spite of this, TMA needs particular pre- and postoperative management in order to avoid the frequent failure rates. A systematic review was undertaken of studies concerning TMA and its care in diabetic foot gangrene. Studies were identified by searching the MEDLINE, Scopus and Science Direct databases until 13 January 2016. All studies were assessed using the Downs and Black quality checklist. Of the 348 records found, 86 matched our inclusion criteria. After reading the full-text articles, we decided to exclude 35 manuscripts because of the following reasons: (1) no innovative or important content, (2) no multivariable analysis, (3) insufficient data, (4) no clear potential biases or strategies to solve them, (5) no clear endpoints and (6) inconsistent or arbitrary conclusions. The final set included 51 articles. In the current literature, there are less data about TMA, indication for the selection of patients, outcomes and complications. Generally, the judgment of an experienced physician is one of the best indicators of subsequent healing. Ankle brachial indices, toe pressures, laser Doppler skin perfusion pressures, angiography and Doppler assessment of foot vasculature may help physicians in this decision. In any case, despite the presumed lower healing rate, it is reasonable to pursue a TMA in a patient with a higher likelihood of continued ambulation. Furthermore, tailored wound closure, adjuvant local treatments and the choice of the most appropriate antibiotic therapy, when infection occurs, are pivotal elements for the success of TMA procedures. TMA is a valuable option for diabetic foot gangrene that can prevent major limb loss and minimise loss of function, thus improving the quality of life for diabetic patients.

From varices to venous ulceration: the story of chronic venous disease described by metalloproteinases.

Chronic venous disease (CVD) and its most frightening complication, chronic venous ulceration (CVU), represent an important socioeconomic burden in the western world. Metalloproteinases have been identified in the pathogenesis of several vascular diseases such as venous problems. The aim of this study was to evaluate a broad range of metalloproteinases, such as matrix metalloproteinases (MMPs), ADAMs (a disintegrin and metalloproteinases) and ADAMTSs (a disintegrin and metalloproteinases with thrombospondin motifs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) and a related protein, neutrophil gelatinase-associated lipocalin (NGAL), in patients with CVD in order to correlate their serum levels with each stage of the disease. We performed a multicenter open-label study that comprised the enrolment of 541 patients with CVD of clinical stages C1-C6, (178 males, 363 females; mean age 57·29, median age 53·72, age range 29-81); 29 subjects without CVD were included in this study (9 males and 20 females; mean age 54·44, median age 50, age range 28-84) as the control group. Enzyme-linked immunosorbent assay (ELISA) was performed for measuring serum levels of proteases and related proteins. The study found that the serum elevation of MMP-2, ADAMTS-1 and ADAMTS-7 appeared to be correlated with the initial stages of CVD, whereas the serum elevation of MMP-1, MMP-8, MMP-9, NGAL, ADAM-10, ADAM-17 and ADAMTS-4 was particularly involved in skin change complications. This study showed that each stage of CVD may be described by particular patterns of metalloproteinases, and this may have therapeutic implications in discovering new targets and new drugs for the treatment of CVD.

Updates in Pathophysiology, Diagnosis and Management of Takayasu Arteritis.

BACKGROUND: Takayasu arteritis (TA) is a rare, systemic, inflammatory vasculitis of granulomatous nature, and still of unknown etiology. It mainly involves the aorta and its major branches and is more commonly seen in women of childbearing age and Asians. TA leads to stenosis, occlusion, or aneurysmal degeneration of large arteries, and its pathogenesis seems to be mainly due to an abnormal cell-mediated immunity, although other molecular and genetic abnormalities may contribute. The diagnosis and treatments lie on clinical and arteriographic findings. Because of its fluctuating course, both clinical scores and biomarkers are currently evaluated. The aim of this review is to report a comprehensive and methodologically robust state of the art about Takayasu arteritis, including the latest data and evidences in the definition, epidemiology, pathogenesis and etiology, clinical manifestations and classification, diagnosis, assessment of disease activity and progression, biomarkers, and treatment. METHODS: We searched all publications addressing definition, epidemiology, pathogenesis, etiology, classification, diagnosis, biomarkers, and treatment of TA. Randomized trials, cohort studies, and reviews were contemplated to give a breadth of clinical data. PubMed and Scopus were searched from August 2010 to November 2015. RESULTS: Of the 3,056 records found, 267 matched our inclusion criteria. After reading the full-text articles, we decided to exclude 169 articles because of the following reasons: (1) no innovative or important content; (2) no multivariable analysis; (3) insufficient data; (4) no clear potential biases or strategies to solve them; (5) no clear end-points; and (6) inconsistent or arbitrary conclusions. The final set included 98 articles. CONCLUSIONS: This review presents the last updates in all fields of Takayasu arteritis. Still today, large areas of TA pathogenesis and disease-activity assessment need to be further investigated to better treat patients with TA.

Symptoms in patients with skin changes due to chronic venous insufficiency often lead to emergency care service: an Italian observational study.

Chronic venous insufficiency (CVI) is the most advanced form of chronic venous disease (CVD), and is often associated with skin changes such as hyperpigmentation, eczema, lipodermatosclerosis and venous skin ulceration that cause discomfort, pain, sleep disturbances, absenteeism in the workplace, disability and deteriorated quality of life (QoL). The purpose of this study is to evaluate the prevalence of CVI and skin changes in patients who turn to Continuous Assistance Services due to the presence of disturbing symptoms of their condition. Data were evaluated by consulting the medical records, during a 16-month period, available with three Continuous Assistance Services of the Italian territory. The overall population of the referring centres consisted of 1186 patients [739 females (62·31%) and 447 males (37·69%)]. Seventy-nine patients (6·66%) consulted the emergency unit for venous symptoms related to CVD. Patients with more severe disease (CVI, categories C4-C6) represented the majority accounting for 60·75%, while patients with moderate disease (C3) accounted for 35·44% and patients with mild disease (C1-C2 stages) accounted for 3·79%. The main finding of this study is that despite CVI not being a disease that commonly requires medical emergency/urgency intervention, patients with CVI, especially in advanced stage with skin changes, may turn to Continuous Assistance Service for treating bothersome symptoms related to their condition.

Trophic effects of polynucleotides and hyaluronic acid in the healing of venous ulcers of the lower limbs: a clinical study.

The aim of this study was to evaluate the results of treatment of venous lower limbs ulcers through the topical application of polynucleotides and hyaluronic acid gel (PNHA): Nucliaskin S™ (Mastelli srl, San Remo, Italy). This study was carried out in 39 consecutive patients who were randomly allocated to two groups: group I (20 patients) received treatment with PNHA (topical gel application two times a week, for a total of 6 weeks); group II (19 patients) received only hyaluronic acid (HA) topical application. All patients received a surgical debridement of the ulcerative lesions before topical treatment with PNHA or HA. Pre-treatment data indicated the area of ulceration. The number of healed ulcers and the variation in area of ulceration were considered as endpoints. The endpoints were observed after 45 days from the beginning of treatment. Complete wound healing occurred in 60% of limbs of group I and in 22% of those of group II patients. The average area reduction was 67% versus 34% in patients of group I and II, respectively. No side effects were recorded in both groups. Our experience shows that PNHA has an elevated trophic effect and speeds the healing rate of venous lower limb ulcers. This treatment may be a valid option in clinical practice.

Effectiveness of prostaglandin E1 in patients with mixed arterial and venous ulcers of the lower limbs.

Mixed arterial and venous ulcers of the lower limbs are present in around 15-30% of patients with chronic venous ulcers (CVUs) and are considered difficult-to-heal wounds. The aim of this study was to evaluate the results of the treatment of mixed arterial and venous ulcers of the lower limbs with prostaglandin E1 (PGE1) infusion. This study was carried out in 48 consecutive patients. Patients who showed intolerability to PGE1, and patients with peripheral neuropathy, blood or systemic diseases, malignancy and acute wound infections or necrotic tissue on the wound bed were excluded. The patients were separated at random into two main groups: group I (25 patients) received standard treatment and PGE1 infusion. Group II (23 patients) received only standard treatment. Pre-treatment data indicated the area of ulceration. The number of healed ulcers and the variation in the area of ulceration were considered as endpoints. The endpoints were noticed after 120 days from the beginning of treatment. Healing occurred in 80% of limbs of group I and in 52·2% of limbs of group II patients. The average reduction in area was 92% versus 60% in patients of group I and II, respectively. During the whole treatment period, the incidence of adverse events was 8% in group I: there was one case of headache and one case of headache and hypotension combined. No side effects were recorded in patients of group II. In conclusion, PGE1 infusion is a determinant in the reduction of the healing time of mixed ulcers of the lower limbs.

Plasma MMP and TIMP evaluation in patients with deep venous thrombosis: could they have a predictive role in the development of post-thrombotic syndrome?

Post-thrombotic syndrome (PTS) is a condition that can develop in about half of the patients with deep vein thrombosis (DVT) of lower limbs. In the present study, we evaluated the expression of inflammatory biomarkers in the early phases of DVT and their correlation with the onset of PTS. Patients were enrolled after the first episode of DVT and were followed up for 1, 4, 8, 12 and 18 months. At each visit, blood sample was collected to evaluate plasma levels of matrix metalloproteinase (MMP)-1,-2,-3,-7,-8 and -9 MMP inhibitors, TIMP-1,-2, neutrophil gelatinase-associated lipocalin (NGAL) and cytokines TNF-α and IL-6. Analysis included 201 patients [86 males (42·79%) and 115 females (57·21%); average age 56 ± 7 years]. Of the 201 patients, 47 (23·38%; 21 males, 26 females) developed PTS during the follow-up period. The control group was made up of 60 individuals without DVT (22 males and 38 females). High plasma levels of MMPs, NGAL and cytokines were recorded during the acute phase after DVT. Moreover, patients with PTS showed higher levels of MMP-1 and MMP-8 with respect to patients without PTS. There is a close relationship between DVT, the individual risk of PTS and specific biomarkers such as MMPs and other related molecules, which may help guide prevention and therapy based on the patient's individual risk profile, and has to be studied in future.

The role of adult tissue-derived stem cells in chronic leg ulcers: a systematic review focused on tissue regeneration medicine.

Wound healing is an articulated process that can be impaired in different steps in chronic wounds. Chronic leg ulcers are a special type of non-healing wounds that represent an important cause of morbidity and public cost in western countries. Because of their common recurrence after conventional managements and increasing prevalence due to an ageing population, newer approaches are needed. Over the last decade, the research has been focused on innovative treatment strategies, including stem-cell-based therapies. After the initial interest in embryonic pluripotent cells, several different types of adult stem cells have been studied because of ethical issues. Specific types of adult stem cells have shown a high potentiality in tissue healing, in both in vitro and in vivo studies. Aim of this review is to clearly report the newest insights on tissue regeneration medicine, with particular regard for chronic leg ulcers.

Study on the efficacy of surgery of the superficial venous system and of compression therapy at early stages of chronic venous disease for the prevention of chronic venous ulceration.

The mainstay of treatment of chronic venous ulceration (CVU), as also suggested by current treatment guidelines for chronic venous disease (CVD), is represented by surgery and compression therapy for which there is strong evidence of their role in clinically relevant improvement in wound healing and also in the reduction of CVU recurrence, but no information is available as to whether or not these treatments provide effective protection from the onset of CVU. In our study, we have followed, for a median time of 13 years, a total of 3947 patients with CVD at classes C2-C3 of CEAP classification, treated with our treatment protocol (surgery and compression therapy) in order to track the natural history of these patients with regards to CVU development. We identified four groups of patients: 2354 patients (59·64%) (Group A) fully adherent to protocols; 848 patients (21·48%) (Group B) fully adherent to surgery and non-compliant to compression therapy; 432 patients (10·95%) (Group C) fully adherent to compression therapy and non-compliant to surgery; and 313 patients (7·93%) (Group D) non-compliant to either treatments. Regardless of compliance to treatments, the ulcer development rates were very similar between groups (range: 3·23-4.79%), with no statistical significance (P = 0·1522). Currents treatments used in the early stages of CVD appear to have no effects to progression to CVU. Additional longitudinal studies are required to confirm these findings.

Biomarkers in post-reperfusion syndrome after acute lower limb ischaemia.

Ischaemia reperfusion (I/R) injury refers to tissue damage caused when blood supply returns to the tissue after a period of ischaemia. Matrix metalloproteinases (MMPs), neutrophil gelatinase-associated lipocalin (NGAL) and cytokines are biomarkers involved in several vascular complications. The aim of this study was to evaluate the role of MMPs, NGAL and inflammatory cytokines in I/R syndrome. We conducted an open label, multicentric, parallel group study, between January 2010 and December 2013. Patients with acute limb ischaemia were enrolled in this study and were divided into two groups: (i) those subjected to fasciotomy and (ii) those not subjected to fasciotomy, according to the onset of compartment syndrome. Plasma and tissue values of MMPs and NGAL as well as plasma cytokines were evaluated. MMPs, NGAL and cytokine levels were higher in patients with compartment syndrome. Biomarkers evaluated in this study may be used in the future as predictors of I/R injury severity and its possible evolution towards post-reperfusion syndrome.

VAC therapy for the treatment of complex wounds after cardio-thoracic surgery.

The aim of this study is to report our experience about the treatment of complex sternal and thoracic wounds following cardiothoracic surgery, using vacuum-assisted closure (VAC therapy. Twelve patients presenting with sternal (five cases) and thoracic (seven cases) wounds that were difficult to heal were treated through VAC therapy after the first surgical debridement. The duration of VAC application ranged from 12 to 36 days with an average hospital stay of 24·6 ± 11·4 days. During a mean follow-up of 12 months, we observed complete wound healing in seven cases (58·3%), in an average period of 25·5 ± 14·3 days; one patient died during follow-up, two patients were lost to follow-up and two patients required definitive surgical closure of the wound cavity. In conclusion, VAC therapy promotes faster wound healing, with shorter hospital stay and subsequent lesser in-hospital cost, reducing the mortality rate in the long run. It also promotes early rehabilitation and alleviates the need for a second procedure, thus improving patient satisfaction, with minimal discomfort or inconvenience.

Spinal cord stimulation to achieve wound healing in a primary lower limb critical ischaemia referral centre.

Critical lower limb ischaemia is a diffuse pathology that could cause claudication, severe ischaemic pain and tissue loss. The common treatment includes modification of risk factors, pharmacological therapy and endovascular or surgical revascularisation of the lower limb to restore a pulsatile flow distally. Spinal cord stimulator is seen as a valid alternative in patients unsuitable for revascularisation after endovascular or surgical revascularisation failure and as adjuvant therapy in the presence of a functioning bypass in patients with extensive tissue loss and gangrene presenting a slow and difficult wound healing. We report our experience on spinal cord stimulation (SCS) indication and implantation in patients with critical lower limb ischaemia, at a high-volume centre for the treatment of peripheral arterial disease.

VAC therapy to promote wound healing after surgical revascularisation for critical lower limb ischaemia.

Vacuum-assisted closure (VAC) therapy is a new emerging non-invasive system in wound care, which speeds up wound healing by causing vacuum, improving tissue perfusion and suctioning the exudates, and facilitating the removal of bacteria from the wound. The application of sub-atmospheric pressure on the lesions seems to alter the cytoskeleton of the cells on the wound bed, triggering a cascade of intracellular signals that increase the rate of cell division and subsequent formation of granulation tissue. The aim of this study is to analyse the results of VAC therapy used as an adjuvant therapy for the treatment of foot wounds in patients affected by critical limb ischaemia (CLI) (Rutherford 6 class) after distal surgical revascularisation, to promote and accelerate the healing of ulcers. Twenty-nine patients (20 males, 9 females; mean age 68·4) affected by CLI of Rutherford 6 class, after surgical revascularisation of the lower limb, underwent VAC therapy in order to speed up wound healing. Complete wound healing was achieved in 19 patients (65·51%), in an average period of 45·4 ± 25·6 days. VAC therapy is a valid aid, after surgical revascularisation, to achieve rapid healing of foot lesions in patients with CLI.

Adjuvant spinal cord stimulation improves wound healing of peripheral tissue loss due to steal syndrome of the hand: clinical challenge treating a difficult case.

Hand ischaemia due to arterial steal syndrome is an infrequent, but potentially serious complication of arteriovenous fistula (AVF) for haemodialysis. We present a case of hand ischaemia caused by steal syndrome in a 69-year-old haemodialysis patient, 10 months after a brachiobasilic fistula creation. The patient underwent multiple operations without resolution of hand pain and tissue loss. The implantation of an adjuvant cervical spinal cord stimulator allowed the patient to obtain complete hand pain relief and wound healing. Probably, the diffuse microangiopathy typical of haemodialysis patients could be responsible for the persistence of ischaemic signs and symptoms after a surgical revascularisation. The effect of sympathetic blockade and the subsequent improvement of the arterial blood flow and tissue oxygenation because of spinal cord stimulation (SCS) can be useful to achieve complete ischaemic pain relief in order to enhance wound healing and to limit the tissue loss. In conclusion, the association of cervical spinal cord stimulation and surgical revascularisation could represent a valid option to treat a critical upper limb ischaemia following steal syndrome due to AVF.

Extreme distal bypass to improve wound healing in Buerger's disease.

Thromboangiitis obliterans or Buerger's disease is a rare non-atherosclerotic segmental inflammatory vasculitis that most commonly involves small and medium-sized arteries, veins and nerves of the extremities, and generally affects young tobacco smokers. A 53-year-old man was found to have critical ischaemia of his left lower limb with foot gangrene. He underwent extremely distal surgical revascularisation using a great saphenous vein bypass graft. The choice of a very distal artery as run-off vessel promoted a faster wound healing and pain relief, with improvement in quality of life.

Matrix metalloproteinases and risk stratification in patients undergoing surgical revascularisation for critical limb ischaemia.

Critical limb ischaemia (CLI) is the most advanced form of peripheral artery disease (PAD) and it is often associated with foot gangrene, which may lead to major amputation of lower limbs, and also with a higher risk of death due to fatal cardiovascular events. Matrix metalloproteinases (MMPs) seem to be involved in atherosclerosis, PAD and CLI. Aim of this study was to evaluate variations in MMP serum levels in patients affected by CLI, before and after lower limb surgical revascularisation through prosthetic or venous bypass. A total of 29 patients (7 females and 22 males, mean age 73·4 years, range 65-83 years) suffering from CLI and submitted to lower extremity bypass (LEB) in our Institution were recruited. Seven patients (group I) underwent LEB using synthetic polytetrafluoroethylene (PTFE) graft material and 22 patients (group II) underwent LEB using autogenous veins. Moreover, 30 healthy age-sex-matched subjects were also enrolled as controls (group III). We documented significantly higher serum MMPs levels (P < 0·01) in patients with CLI (groups I and II) with respect to control group (group III). Finally, five patients with CLI (17·2%) showed poor outcomes (major amputations or death), and enzyme-linked immunosorbent assay (ELISA) test showed very high levels of MMP-1 and MMP-8. MMP serum levels seem to be able to predict the clinical outcomes of patients with CLI.

Effects of a new nutraceutical substance on clinical and molecular parameters in patients with chronic venous ulceration.

Pathophysiological events involved in the onset of chronic venous ulceration (CVU) are inflammation, activation of polymorphonucleates (PMNs) and secretion of proteases such as matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) that is a support for vascular and tissutal wall. MMPs, neutrophil gelatinase-associated lipocalin (NGAL) and inflammatory cytokines are overexpressed in CVUs and they could play a central role in pathophysiological mechanisms of skin lesion and delayed wound healing. Bioflavonoids, such as diosmin and other compounds, appear to have several provessel function activities including anti-inflammatory, antioxidant and phlebotonic effects and are widely used in the treatment of chronic venous disease (CVD)-related problems. In this article, we evaluated the effects of Axaven(®) , a new nutraceutical on both clinical and molecular parameters in patients with CVUs. During the study period, 83 patients with CVUs of both sexes were enrolled and divided into two groups: group A (treated group): 25 females and 19 males (median age is 67·7 years) received standard treatment (compression therapy and surgical correction of superficial venous incompetence) + Axaven(®) once a day for 8 months as adjunctive treatment. Group B (control group): 24 females and 15 males (median age is 65·2 years) were treated only with basic treatment according to their clinical conditions. In our study, the administration of Axaven(®) in patients with CVUs was able to decrease inflammatory cytokines, MMPs and NGAL, inducing an improvement of both symptoms with an increase of the speed of wound healing.

Absorbable suture material in carotid surgery.

BACKGROUND: Absorbable sutures are not generally accepted by most vascular surgeons for the fear of breakage of the suture line and the risk of aneurysmal formation, except in cases of paediatric surgery or in case of infections. Aim of this study is to provide evidence of safety and efficacy of the use of absorbable suture materials in carotid surgery. PATIENTS AND METHODS: In an 11 year period, 1126 patients (659 male [58.5%], 467 female [41.5%], median age 72) underwent carotid endarterectomy for carotid stenosis by either conventional with primary closure (cCEA) or eversion (eCEA) techniques. Patients were randomised into two groups according to the type of suture material used. In Group A, absorbable suture material (polyglycolic acid) was used and in Group B non-absorbable suture material (polypropylene) was used. Primary end-point was to compare severe restenosis and aneurysmal formation rates between the two groups of patients. For statistical analysis only cases with a minimum period of follow-up of 12 months were considered. RESULTS: A total of 868 surgical procedures were considered for data analysis. Median follow-up was 6 years (range 1-10 years). The rate of postoperative complications was better for group A for both cCEA and eCEA procedures: 3.5% and 2.0% for group A, respectively, and 11.8% and 12.9% for group B, respectively. CONCLUSIONS: In carotid surgery, the use of absorbable suture material seems to be safe and effective and with a general lower complications rate compared to the use of non-absorbable materials.

Concomitant aortic leiomyosarcoma and takayasu arteritis in a 55-year-old male patient.

Takayasu arteritis is a form of large vessel granulomatous vasculitis affecting often young or middle-aged women, especially of Asian descent. It mainly affects the aorta and its branches. Primary malignancies, such as leiomyosarcoma of the aorta are extremely rare. This report discusses the exceptional and concomitant association of Takayasu arteritis and aortic leiomyosarcoma in a 55-year-old male. The patient suffered from systemic signs and symptoms related to arteritis together with claudication of left upper limb due to left artery subclavian stenosis. After instrumental evaluation, an infiltrating neoplasm at the level of abdominal aortic wall was detected and the patient underwent en bloc excision of the mass together with abdominal aorta and subsequent aortoaortic by pass reconstruction. Histologic findings showed an arterial leiomyosarcoma combined with elements of arterial inflammation. Patients completed therapeutic scheme with chemotherapy (doxorubicin and isosfamide) and radiotherapy for the cancer condition, as well as medical treatment (prednisone and adalimumab) for Takayasu arteritis. After 12 months, the patient showed no cancer recurrence and complete normalization of inflammatory indexes and symptoms of Takayasu arteritis.

Hemorrhoids and matrix metalloproteinases: A multicenter study on the predictive role of biomarkers.

INTRODUCTION: An association between hemorrhoidal disease and matrix metalloproteinases (MMPs) has been described previously. MMPs regulate extracellular structural proteins and tissue remodeling. Neutrophil gelatinase-associated lipocalin (NGAL) is involved in the regulation of MMP activity. The aim of this work was to study the relationship between tissue immunoreactive levels of MMPs and NGAL and different stages of hemorrhoids. METHODS: In a multicenter, open-label, prospective study, the population under investigation consisted of 2 groups: group I (with symptomatic hemorrhoids; Goligher grade I-IV) and group II (healthy volunteers). RESULTS: We enrolled 97 patients with hemorrhoids: 21 with grade I hemorrhoids, 37 with grade II, 14 with grade III, and 25 with grade IV. Finally, 90 healthy volunteers (53 males and 37 females; age range, 19-70 years; median, 56) were enrolled in group II. Enzyme-linked immunosorbent assay and Western blot analysis revealed greater levels of immunoreactive MMPs and NGAL in all patients with hemorrhoids. We recorded significantly greater levels of MMP-1 and MMP-3 in grade I and II patients compared with control, and greater levels of MMP-3, MMP-7, MMP-8, and MMP-9 in grade III compared with grade II. MMP-9 and NGAL were particularly increased in patients with grade IV especially in case of thrombosed hemorrhoids. CONCLUSION: These results provide potentially important insights into the understanding of the natural history of hemorrhoids. MMPs and NGAL play a role in development of disease and may represent molecular markers for the complications such as hemorrhoidal thrombosis.