Coagulation Thrombosis Research Articles

PURIFICATION, CHARACTERIZATION, AND EVALUATION OF FIBRINOLYTIC ACTIVITY OF STAPHYLOKINASE FROM LOCALLY ISOLATED STAPHYLOCOCCUS AUREUS GH38

This study was aimed to purify, characteristic, and fibrinolytic activity of staphylokinase (SAK), is an enzyme activates plasminogen to form plasmin, which digest fibrin clots that cause thrombosis clot. Staphylokinase was purified from local isolate Staphylococcus aureus GH38 by ammonium sulfate precipitation at 70% saturation followed by ion exchange chromatography (CM-Cellulose) with a purification fold 2.73, and 1.53, and recovery 72.1, and 33.11% in wash and elution steps respectively. The partially purified enzyme was high activity at 40°C with pH 7 and the enzyme retained 100% of its activity at 35°C with pH 7. The activity of an enzyme increased by its treatment with calcium and sodium chloride, while the activity affected when incubated with mercury, silver, and iron chloride. The enzyme have high effective against thrombus (blood clot), which encourage the use of enzyme in the treatment as therapeutic agent to remove clots formed in the human body.

Understanding Inflammatory Responses in the Manifestation of Prothrombotic Phenotypes.

Inflammasome complex is a multimeric protein comprising of upstream sensor protein of nucleotide-binding oligomerization domain (NOD)-like receptor family. It has an adaptor protein apoptosis-associated speck-like protein and downstream effector cysteine protease procaspase-1. Activation of inflammasome complex is body’s innate response to pathogen attack but its abnormal activation results in many inflammatory and cardiovascular disorders including thrombosis. It has displayed a prominent role in the clot formation advocating an interplay between inflammation and coagulation cascades. Therefore, elucidation of inflammasome and its molecular mechanisms in the manifestation of prothrombotic phenotypes becomes pertinent. Thrombosis is the formation and propagation of blood clot in the arterial or venous system due to several interactions of vascular and immune factors. It is a prevalent pathology underlying disorders like venous thromboembolism, stroke and acute coronary syndrome; thus, making thrombosis, a major contributor to the global disease burden. Recently studies have established a strong connection of inflammatory processes with this blood coagulation disorder. The hemostatic balance in thrombosis gets altered by the inflammatory mechanisms resulting in endothelial and platelet activation that subsequently increases secretion of several prothrombotic and antifibrinolytic factors. The upregulation of these factors is the critical event in the pathogenesis of thrombosis. Among various inflammasome, nucleotide-binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) is one of the best-studied sterile inflammasome strengthening a link between inflammation and coagulation in thrombosis. NLRP3 activation results in the catalytic conversion of procaspase-1 to active caspase-1, which facilitate the maturation of interleukin-1β (IL-1β) and interleukin-18. These cytokines are responsible for immune cells activation critical for immune responses. These responses further results in endothelial and platelet activation and aggregation. However, the exact molecular mechanism related to the pathogenesis of thrombosis is still elusive. There have been several reports that demonstrate Tissue factor (TF)-mediated signaling in the production of pro-inflammatory cytokines enhancing inflammation by activating protease-activated receptors on various cells, which lead to additional cytokine expression. Therefore, it would be illuminating to interpret the inflammasomes regulation in coagulation and inflammation. This review, thus, tries to comprehensively compile emerging regulatory roles of the inflammasomes in thrombosis and discusses their molecular pathways in the manifestation of thrombotic phenotypes.

Protection from Psoriasis-Related Thrombosis after Inhibition of IL-23 or IL-17A

Psoriasis patients experience chronic systemic skin inflammation and develop cardiovascular comorbidities that shorten their lifespan. Whether cardiovascular disease is improved by treatment with current biologics that target disease-specific pathways is unclear. KC-Tie2 mice develop psoriasiform skin inflammation with increases in IL-23 and IL-17A and proinflammatory monocytosis and neutrophilia that precedes development of carotid artery thrombus formation. To examine whether targeted blockade of IL-23 or IL-17A in KC-Tie2 psoriasis mice improves cardiovascular outcomes, mice were treated systemically for 6 weeks with antibodies targeting IL-17A, IL-17RA, IL-12/23p40, or IL-23p19. Skin inflammation; thrombosis clotting times; and percentage of splenic monocytes, neutrophils, and CD4 T cells were examined. Skin inflammation significantly improved in KC-Tie2 mice treated with each of the antibodies targeting IL-23, IL-17A, or IL-17RA, consistent with clinical efficacy observed in psoriasis patients. The time to occlusive thrombus formation lengthened in these mice and correlated with attenuated acanthosis. This decrease in skin inflammation paralleled decreases in splenic neutrophils (CD11b+Ly6G+) but not monocytes (CD11b+Ly6Chigh) or T cells (CD4+). Our data show that targeted inhibition of IL-23 or IL-17A improves psoriasis-like skin disease and also improves cardiovascular disease in mice.

Coordinated Membrane Ballooning and Procoagulant Spreading in Human Platelets.

Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Chronic Skin-Specific Inflammation Promotes Vascular Inflammation and Thrombosis

Patients with psoriasis have systemic and vascular inflammation and are at increased risk for myocardial infarction, stroke, and cardiovascular death. However, the underlying mechanism(s) mediating the link between psoriasis and vascular disease is incompletely defined. This study sought to determine whether chronic skin-specific inflammation has the capacity to promote vascular inflammation and thrombosis. Using the KC-Tie2 doxycycline-repressible (Dox-off) murine model of psoriasiform skin disease, spontaneous aortic root inflammation was observed in 33% of KC-Tie2 compared to 0% of control mice by 12 months of age (P=0.04) and was characterized by the accumulation of macrophages, T-lymphocytes and B-lymphocytes and reduced collagen content and increased elastin breaks. Importantly, aortic inflammation was preceded by increases in serum TNF-α, IL-17A, VEGF, IL-12, MCP-1 and S100A8/A9 as well as splenic and circulating CD11b+Ly-6Chi pro-inflammatory monocytes. Doxycycline treatment of old mice with severe skin disease eliminated skin inflammation and aortic root lesion presence in 1 year old KC-Tie2 animals. Given the bi-directional link between inflammation and thrombosis, arterial thrombosis was assessed in KC-Tie2 and control mice; mean time to occlusive thrombus formation was shortened by 64% (P=0.002) in KC-Tie2 animals; doxycycline treatment returned thrombosis clotting times to control mouse levels (P=0.69). These findings demonstrate that sustained skin-specific inflammation promotes aortic root inflammation and thrombosis and suggest that aggressive treatment of skin inflammation may attenuate pro-inflammatory and prothrombotic pathways that produce cardiovascular disease in psoriasis patients.

Acute Iliac Stent Thrombosis – an Unusual Complication Following Use of the Lloyd-Davies Position

Introduction left-sided claudication and left foot paraesthesia. Clinical examination revealed that all left lower limb Over recent years endovascular stenting for iliac artery pulses were absent but there was no sensori-motor deocclusive disease has become a well accepted treatficit, muscle tenderness or swelling. Urgent angioment, with good long-term patency. As many patients graphy confirmed thrombosis of the left CIA stent (Fig. with stents are elderly, it is not uncommon for them 1A). Regionally directed thrombolysis (rt-PA) was comto require other surgical procedures. We present the menced, but after 17 h check angiography showed no case of a 62-year-old who developed acute thrombosis clot dissolution. He therefore underwent emergency of his common iliac artery (CIA) stent following transthrombectomy in the angiography suite, under general urethral resection of a bladder tumour (TURBT) peranaesthesia. Following surgical cut-down to the left formed in the Lloyd-Davies position. This is previously common femoral artery, the occluded CIA stent was unreported in the literature. traversed with a combination of a 0.035′′ straight guidewire (1′′ tip, Cook, U.K.) and a Cobra catheter (Cordis, U.K.). The guidewire was exchanged for a 0.014′′ wire Case Report and the catheter removed. Following arteriotomy, balloon thrombectomy was performed using a fogarty A 62-year-old man was admitted to another hospital embolectomy catheter (Fogarty Thru-Lumen Emfor TURBT. He was a known arteriopath and had bolectomy Catheter, Baxter, CA, U.S.A.). Completion undergone deployment of a left CIA nitinol stent angiography showed a widely patent stent with ex(Memotherm, Angiomed, Bard, U.K.) for intermittent cellent run-off (Fig. 1B). Following this angioplasty was claudication. This had been performed two years preperformed with an 8 mm angioplasty balloon (Smash, viously and serial duplex surveillance showed no eviBoston Scientific) to ensure that no restenosis had been dence of restenosis. TURBT was performed under missed but the balloon inflated fully without any regeneral anaesthetic in the Lloyd-Davies position. The sistance. The patient was discharged after 5 days and procedure lasted 15 min, without intra-operative hyporemains well 6 months later without incident. tension. In recovery the patient complained of left leg pain and paraesthesia and this was treated with analgesics. Despite failing to resolve completely, the pain Discussion was assumed to be musculo–skeletel and the patient was discharged the following day. Endovascular stents are at risk of fracture when subject After 3 weeks he presented to us with short distance to ‘‘unusual’’ compressive or flexion forces and this has been documented in the carotid, subclavian and femoro–popliteal arteries. Common iliac stents lie at ∗ Please address all correspondence to: D. J. A. Scott, Department of the level of the pelvic brim and should not be subject Vascular Surgery and Vascular Interventional Radiology, St. James’s University Teaching Hospital, Beckett Street, Leeds LS9 7TF, U.K. to such forces. Despite this iliac stent thrombosis may

High Plasma Homocysteine: A Risk Factor for Arterial and Venous Thrombosis in Patients with Normal Coagulation Profiles

A high plasma homocysteine concentration is associated with premature vascular disease and thrombosis. The association between high homocysteine concentrations and thrombosis in patients with a normal coagulation profile is unknown. Sixty adults (37 men and 23 women, mean age 46 years) with documented thrombosis were compared with age-and sex-matched controls. Those with risk factors for thrombosis or abnormal coagulation profiles were excluded. Homocysteine concentrations were higher in cases than controls (21.8 ± 13.8 vs 11.0 ± 4.7 μmol/L, p < 0.001). A cut point for defining high homocysteine concentrations was determined at 13 μmol/L and conferred an increased odds ratio for thrombosis overall (7.8, 95% CI 3.0-20.2, p < 0.001) as well as in men (8.9, 95% CI 3.0-26.1; p < 0.001) and women (37.8, 95% CI 6.5-213.9; p < 0.01). A high plasma homocysteine is a risk factor for thrombosis in patients with a normal coagulation profile. This common abnormality should be sought in patients with otherwise unexplained thrombotic episodes. Key Words: Homocysteine—Thrombosis—Clotting.