Arteriolar Thrombosis Research Articles

An updated classification of thrombotic microangiopathies and treatment of complement gene variant-mediated thrombotic microangiopathy.

Conditions presenting with signs of thrombotic microangiopathies (TMAs) comprise a wide spectrum of different diseases. While pathological hallmarks are thrombosis of arterioles and capillaries, clinical signs are mechanical haemolysis, thrombocytopenia and acute renal injury or neurological manifestations. The current classification of various syndromes of TMA is heterogeneous and often does not take the underlying pathophysiology into consideration. Therefore we propose a simplified classification based on the aetiology of different syndromes leading to TMA. We propose to categorize different TMA syndromes in hereditary and acquired forms and classify them based on the genetic background or underlying conditions. Of course, this classification is not always distinctly applicable in each case and from time to time reassessment of the established diagnosis is strongly recommended. The recommended treatment of TMA in the past was plasma exchange (PE). However, recently, the terminal complement inhibitor eculizumab became commercially available and has shown promising results in different open-label studies and case series. In our centre, first-line therapy is PE; however, patients are instantly switched to complement inhibitory therapy in case of treatment failure or intolerance.

Unusual Case of Simultaneous Acute Hepatitis and Acute Pancreatitis in a Bodybuilder

The use of anabolic steroids is widespread, particularly among bodybuilders. Most athletes have only a crude pharmacological knowledge regarding these drugs and warnings of steroid misuse are neglected. The illicit use of Androgenic Anabolic Steroids (AAS) to obtain an athletic, healthy looking body can lead to serious and often irreversible organ damage [1]. Anabolic steroids with 17 alpha carbon substitutions have been associated with a cholestatic injury with little hepatocellular injury. In the case of hepatoxicity and severe cholestasis the prompt withdrawal of the steroid and the administration of ursodeoxycholic acid are recommended [2]. Steroid also is known to cause acute pancreatitis which would result in acute onset abdominal pain and vomiting. Possible mechanisms for drug-induced pancreatitis include immune-mediated inflammatory response, direct cellular toxicity, arteriolar thrombosis, and metabolic effects.

Non-uraemic calciphylaxis following rapid weight loss – a case report

Background: Calciphylaxis is a well-known complication in end-stage kidney disease (ESKD) and haemodialysis, and is characterised by cutaneous ischaemia secondary to arteriolar calcification and thrombosis.1,2.In contrast, literature describing calciphylaxis in the absence of ESKD remains scarce. Possible causes of non-uraemic calciphylaxis include hyperparathyroidism, alcoholic liver diseases and weight loss. We present a case of non-uraemic calciphylaxis following rapid intentional weight loss, and discuss the clinical and histopathological status.

Platelet Shp2 negatively regulates thrombus stability under high shear stress

Essentials Shp2 negatively regulates thrombus stability under pathological shear rate. Shp2 suppresses TXA2 receptor-mediated platelet dense granule secretion. Through αIIbβ3 outside-in signaling, Shp2 targets calmodulin-dependent activation of Akt. Shp2 may serve to prevent the formation of unwanted occlusive thrombi. SUMMARY: Background Perpetuation is the final phase of thrombus formation; however, its mechanisms and regulation are poorly understood. Objective To investigate the mechanism of Shp2 in platelet function and thrombosis. Methods and results We demonstrate that the platelet-expressed Src homology region2 domain-containing protein tyrosine phosphatase Shp2 is a negative regulator of thrombus stability under high shear stress. In a ferric chloride-induced mesenteric arteriole thrombosis model, megakaryocyte/platelet-specific Shp2-deficient mice showed less thrombi shedding than wild-type mice, although their occlusion times were comparable. In accordance with this invivo phenotype, a microfluidic whole-blood perfusion assay revealed that the thrombi formed on collagen surfaces by Shp2-deficient platelets were more stable under high shear rates than those produced by wild-type platelets. Whereas Shp2 deficiency did not alter platelet responsiveness towards thrombin, ADP and collagen stimulation, Shp2-deficient platelets showed increased dense granule secretion when stimulated by the thromboxaneA2 analog U46619. Shp2 appears to act downstream of integrin αIIb β3 outside-in signaling, inhibiting the phosphorylation of Akt (Ser473 and Thr308) and dense granule secretion. Calmodulin was also shown tobind both Shp2 and Akt, linking Shp2 to Akt activation. Conclusions Platelet Shp2 negatively regulates thrombus perpetuation under high shear stress.This signaling pathway may constitute an important mechanism for the prevention of unwanted occlusive thrombus formation, without dramatically interfering with hemostasis.

Study of Osteocyte Behavior by High-Resolution Intravital Imaging Following Photo-Induced Ischemia

Ischemic injuries and local hypoxia can result in osteocytes dysfunction and play a key role in the pathogenesis of avascular osteonecrosis. Conventional imaging techniques including magnetic resonance imaging (MRI) and computed tomography (CT) can reveal structural and functional changes within bony anatomy; however, characterization of osteocyte behavioral dynamics in the setting of osteonecrosis at the single cell resolution is limited. Here, we demonstrate an optical approach to study real-time osteocyte functions in vivo. Using nicotinamide adenine dinucleotide (NADH) as a biomarker for metabolic dynamics in osteocytes, we showed that NADH level within osteocytes transiently increase significantly after local ischemia through non-invasive photo-induced thrombosis of afferent arterioles followed by a steady decline. Our study presents a non-invasive optical approach to study osteocyte behavior through the modulation of local environmental conditions. Thus it provides a powerful toolkit to study cellular processes involved in bone pathologies in vivo.

Venous Thrombophilia, Platelet von Willebrand Factor Mediated Arteriolar Microvascular Thrombosis in JAK2V617F Mutated Thrombocythemia and Acquired ADAMTS13 Deficiency as Causes of Intrahepatic Obstructive Microvascular Liver Diseases in Budd-Chiari Syndrome and Splanchnic Vein Thrombosis

Congenital venous thrombophilia is associated with increased risk of venous thrombosis at adolescent and adult age, with recurrent abortions and fetal loss in females, and less frequently with splanchnic vein thrombosis, but not with arteriolar microvascular circulation disturbances

Intrarenal Arterial Lesions Are Associated with Higher Blood Pressure, Reduced Renal Function and Poorer Renal Outcomes in Patients with IgA Nephropathy

Background/Aims: Arterial fibrotic intimal thickening and arteriolar hyaline are considered common pathological features in immunoglobulin A nephropathy (IgAN), whereas little is known about the acute pathological manifestations of endothelial cell injury. The aim of this study was to investigate characteristics of intrarenal arterial lesions and to estimate their prognostic values in patients with IgAN. The primary renal endpoint was a 50% reduction in estimated glomerular filtration rate (eGFR) or end-stage renal disease (ESRD). Methods: Various renal arterial lesions (arterial fibrotic intimal thickening, arteriolar hyaline, arteriolar endotheliocyte swelling, arteriolar inflammatory cell infiltration, and arteriolar thrombosis) in 1683 patients with IgAN were reviewed and reclassified using a semi-quantitative scoring system. Their correlations with clinical features, pathological characteristics, and renal outcomes were evaluated. Results: The prevalence of intrarenal arterial lesions was up to 72.2% in IgAN patients. There were 978 patients (58.1%) with arterial fibrotic intimal thickening, 350 patients (20.8%) with arteriolar hyaline, 432 patients (25.7%) with arteriolar endotheliocyte swelling, 356 patients (21.2%) with arteriolar inflammatory cell infiltration and 43 patients (2.6%) with arteriolar thrombosis. Arterial fibrotic intimal thickening and arteriolar hyaline were strongly associated with higher mean arterial pressure (MAP) and reduced eGFR (P < 0.001) but were not related to proteinuria at the time of renal biopsy. In contrast, arteriolar endotheliocyte swelling and arteriolar thrombosis were correlated with heavier proteinuria as well as higher MAP and reduced eGFR. During follow-up, patients with vascular lesions received more renin-angiotensin system (RAS) blockade and less glucocorticoid and showed poorer renal outcomes. Univariate Cox model showed that the presence of renal vascular lesions [hazard ratio (HR) = 25.01, 95% confidence interval (CI): 6.19 to 101.03, P < 0.001] was a risk factor for renal outcomes. However, in multivariable Cox analysis, which included clinical factors and the Oxford-MEST-C, vascular lesions were not significantly associated with an increased risk of renal failure. Remarkably, the impact of vascular lesions on the survival from ESRD or 50% reduction in renal function was eliminated by the use of RAS blockade after adjustment for eGFR, proteinuria, and MAP. Conclusion: Our study demonstrates the high prevalence of vascular lesions, including the chronic and acute arterial pathological changes, in patients with IgAN. The presence of vascular lesions is associated with higher MAP, reduced eGFR and poorer renal outcomes, which could be influenced by the RAS blockade treatment.

Main Factors Influencing FeCl3-Induced Mouse Mesenteric Arteriole Thrombosis Model

To investigate the factors that influence FeCl3-induced mouse mesenteric arteriole thrombosis model. Platelets were isolated from donor mice and labeled with Calcein-AM. Mice were transfused intravenously with Calcein-AM labeled platelets. The influence of mouse ages (3-6 weeks, 6-10 weeks and >10 weeks), transfused platelets counts (1×107, 1×108 and 2×108 platelets) and concentrations of FeCl3 (6%, 12%, 24% and 48%) on FeCl3-induced thrombosis model were compared. The occlusion time was 16 min for mice aged 3-6 weeks, which was shorter than that for 6 mice aged 6-10 weeks(25 min)(P<0.05) and that for mice aged >10 weeks(38 min)(P<0.01). The occlusion time resulting from transfusion of 1×108 and 2×108 of pletclets was 15-18 min, which was shorter than that of transfusion 1×107 platelets (30 mins). The occlusion time resulting from transfusion of 6% and 12% FeCl3 was from 15 to 20 min, however the transfusion of 24% and 48% FeCl3 all in all leads to vessel occlusion within 10 min. The factors influencing the success of FeCl3-induced mouse thrombosis model are more. Transfusion of 1×108 to 2×108 labeled platelets to 3-6 week-old mice, and 6% to 12% of FeCl3 should be used to induce thrombosis, and the experimental conditions should be optimized for this animal model, therefore, it is easier for us to set up a mouse mesenteric arteriole thrombosis model.

Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase.

Class III phosphoinositide 3-kinase, also known as VPS34 (vacuolar protein sorting 34), is a highly conserved enzyme regulating important cellular functions such as NADPH oxidase (NOX) assembly, membrane trafficking, and autophagy. Although VPS34 is expressed in platelets, its involvement in platelet activation remains unclear. Herein, we investigated the role of VPS34 in platelet activation and thrombus formation using VPS34 knockout mice. Platelet-specific VPS34-deficient mice were generated and characterized. VPS34 deficiency in platelets did not influence tail bleeding time. In a ferric chloride-induced mesenteric arteriolar thrombosis model, VPS34-/- mice exhibited a prolonged vessel occlusion time compared with wild-type mice (42.05±4.09 versus 18.30±2.47 minutes). In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on collagen under arterial shear was significantly reduced for VPS34-/- platelets. VPS34-/- platelets displayed an impaired aggregation and dense granule secretion in response to low doses of collagen or thrombin. VPS34 deficiency delayed clot retraction but did not influence platelet spreading on fibrinogen. We also demonstrated that VPS34 deficiency altered the basal level of autophagy in resting platelets and hampered NOX assembly and mTOR (mammalian target of rapamycin) signaling during platelet activation. Importantly, we identified the NOX-dependent reactive oxygen species generation as the major downstream effector of VPS34, which in turn can mediate platelet activation. In addition, by using a specific inhibitor 3-methyladenine, VPS34 was found to operate through a similar NOX-dependent mechanism to promote human platelet activation. Platelet VPS34 is critical for thrombosis but dispensable for hemostasis. VPS34 regulates platelet activation by influencing NOX assembly.

Direct oral anticoagulant medications in calciphylaxis.

Recent studies suggest that calciphylaxis is a thrombotic condition in which arteriolar thrombosis leads to painful skin infarcts and consequent morbidity and mortality. Paradoxically, warfarin is implicated as a risk factor for calciphylaxis. Our objective is to report the use of oral direct thrombin and factor Xa inhibitors (termed direct oral anticoagulants [DOACs]) in patients with calciphylaxis. We retrospectively reviewed records of 16 patients with calciphylaxis who received concomitant administration of novel anticoagulants. Patient data, including demographics, comorbidities, other treatments, and adverse events, were abstracted from the health records. Eleven patients (69%) had chronic kidney disease (stage ≥3A), and eight (50%) received dialysis. Apixaban was the most frequently used agent (n = 11 [69%]). Dabigatran (n = 4 [25%]) and rivaroxaban (n = 2 [13%]) were reserved for patients with mild renal impairment (stage ≤2). One clinically relevant but nonmajor bleeding event occurred. There were no major bleeding events. Nine patients (56%) were alive at last follow-up, and five (31%) had complete resolution of their calciphylaxis (mean follow-up, 523 days; range, 26-1884 days). DOACs were safe and well tolerated in patients with calciphylaxis, in this initial experience. Several patients had improvement or resolution of calciphylaxis in response to therapy that included DOACs. The degree of renal impairment should guide DOAC choice. Randomized trials are required to determine treatment efficacy.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y12 receptor expression in megakaryocytes. Platelet P2Y12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y12 expression correlates with ADP-induced platelet aggregation (r=0.89, P<0.01). P2Y12 in platelets from patients with diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P<0.05). Using a FeCl3-injury mesenteric arteriole thrombosis model in rats and an arteriovenous shunt thrombosis model in rats, we found that the inverse agonist AR-C78511 has greater antithrombotic effects on GK rats with diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P<0.01). We also found that a pathway involving high glucose-reactive oxygen species-nuclear factor-κB increases platelet P2Y12 receptor expression in diabetes mellitus. Platelet P2Y12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus.

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis.

Adequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis. ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl3-induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy.

Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis ex vivo , supporting a key role for 12-LOX in regulation of in vivo thrombosis. While pharmacologically targeting 12-LOX in vivo has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic in vivo was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation ex vivo in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin. In vivo , PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl 3 -induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX -/- mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX -/- mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.

Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis

Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3-/-) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3-/- bone marrow-derived cells had longer occlusion times than RIP3-/- mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3-/- platelets. Moreover, RIP3 interacted with Gα13 Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

Abstract 54: Differential Regulation of NADPH-oxidases 1 and 2 and a Common Syk/PLC/Calcium-dependent ROS Signaling Pathway Mediating Platelet Activation

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. This study investigates how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) mediates different platelet activation pathways. Approach and Results: ROS generation in different platelet activation pathways are mediated differentially by NOX1 and NOX2. NOX1 -/y platelets showed no defects in platelet aggregation and secretion induced by glycoprotein (GP) VI agonists, collagen-related peptide (CRP), but were partially defective in platelet aggregation and secretion induced by low doses of agonists of G protein coupled receptor (GPCR), thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619. In contrast, NOX2 -/- platelets showed significantly defective platelet aggregation and secretion induced by CRP, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1 -/- platelets stimulated with thrombin, but not CRP, whereas NOX2 -/- platelets were defective ROS generation induced by CRP or thrombin. These differential effects of NOX1 and NOX2 is likely due to upstream differential regulation of these different enzymes, as thrombin-stimulated NOX1-/y platelets and CRP-stimulated NOX2-/- platelets similarly showed defective activation of tyrosine kinase Syk, its downstream target phospholipase Cγ (PLCγ) and calcium mobilization, which is mediated by PLC. Furthermore, mitogen-activated protein kinase pathways, which is another important platelet activation pathway was not significantly affected in either NOX1-/y or NOX2-/- platelets. Finally, NOX-/- platelets is defective in mediating arteriolar thrombosis in vivo, although minimally affected tail bleeding time. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways. The differential roles of these enzyme are due to differential upstream regulation. Both NOX isoforms mediates platelet activation via a common ROS-dependent activation Src-PLC-calcium signaling pathway.

Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation in vivo

Background: Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of in vivo thrombosis. The ability to target 12-LOX in vivo has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation in vivo using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics. Methods: ML355 effects on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber, and in vivo by thrombus formation and vessel occlusion in small and large vessels in 12-LOX -/- , WT mice, and mice treated with ML355 via intravital microscopy using the FeCl 3 and laser injury models. Results: In in vitro platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX -/- mice were impaired in both laser and FeCl 3 -induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX -/- mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists. Ex vivo high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion in vivo . Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.

Arterial thrombosis in the context of HCV-associated vascular disease can be prevented by protein C.

Hepatitis C virus (HCV) infection is a major problem worldwide. HCV is not limited to liver disease but is frequently complicated by immune-mediated extrahepatic manifestations such as glomerulonephritis or vasculitis. A fatal complication of HCV-associated vascular disease is thrombosis. Polyriboinosinic:polyribocytidylic acid (poly (I:C)), a synthetic analog of viral RNA, induces a Toll-like receptor 3 (TLR3)-dependent arteriolar thrombosis without significant thrombus formation in venules in vivo. These procoagulant effects are caused by increased endothelial synthesis of tissue factor and PAI-1 without platelet activation. In addition to human umbilical endothelial cells (HUVEC), human mesangial cells (HMC) produce procoagulatory factors, cytokines and adhesion molecules after stimulation with poly (I:C) or HCV-containing cryoprecipitates from a patient with a HCV infection as well. Activated protein C (APC) is able to prevent the induction of procoagulatory factors in HUVEC and HMC in vitro and blocks the effects of poly (I:C) and HCV-RNA on the expression of cytokines and adhesion molecules in HMC but not in HUVEC. In vivo, protein C inhibits poly (I:C)-induced arteriolar thrombosis. Thus, endothelial cells are de facto able to actively participate in immune-mediated vascular thrombosis caused by viral infections. Finally, we provide evidence for the ability of protein C to inhibit TLR3-mediated arteriolar thrombosis caused by HCV infection.

Calciphylaxis in chronic kidney disease

PurposeCalciphylaxis or Calcific Uraemic Arteriolopathy (CUA) is a rare systemic disorder associated with endstage renal disease and carrying a very poor prognosis and high mortality. It is characterized by skin necrosis and gangrene secondary to thrombosis and calcification of dermo-epidermal arterioles. Pathogenetic mechanisms inducing calciphylaxis are for the most part unknown.MethodsWe report a case of calciphylaxis in a patient on long-term haemodialysis with severe secondary hyperparathyroidism. Despite therapies for hyperparathyroidism, the injury healed only after parathyroidectomy.ResultsCalciphylaxis is an obliterative vasculopathy. The role of severe hyperparathyroidism in the pathogenesis of calciphylaxis is not well understood. A unique feature is the phenotypic differentiation of smooth muscle cells into osteoid-like cells. The vascular smooth muscle cells, exposed to high inorganic phosphate levels, could be transformed into osteoblast-like cells in patients undergoing haemodialysis with a...

Misshapen/NIK-related kinase (MINK1) is involved in platelet function, hemostasis, and thrombus formation

AbstractThe sterile-20 kinase misshapen/Nck-interacting kinase (NIK)–related kinase 1 (MINK1) is involved in many important cellular processes such as growth, cytoskeletal rearrangement, and motility. Here, with MINK1-deficient (MINK1−/−) mice, we showed that MINK1 plays an important role in hemostasis and thrombosis via the regulation of platelet functions. In the tail-bleeding assay, MINK1−/− mice exhibited a longer bleeding time than wild-type (WT) mice (575.2 ± 59.7 seconds vs 419.6 ± 66.9 seconds). In a model of ferric chloride–induced mesenteric arteriolar thrombosis, vessel occlusion times were twice as long in MINK1−/− mice as in WT mice. In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on a collagen matrix under arterial shear conditions was significantly reduced in MINK1−/− platelets. Moreover, MINK1−/− platelets demonstrated impaired aggregation and secretion in response to low doses of thrombin and collagen. Furthermore, platelet spreading on fibrinogen was largely hampered in MINK1−/− platelets. The functional differences of MINK1−/− platelets could be attributed to impaired adenosine 5′-diphosphate secretion. Signaling events associated with MINK1 appeared to involve extracellular signal-regulated kinase, p38, and Akt. Hence, MINK1 may be an important signaling molecule that mediates mitogen-activated protein kinase signaling and participates in platelet activation and thrombus formation.

Update on leukoreduction therapies as alternatives for the treatment of severe pertussis

Pertussis is a contagious disease that in patients under 1 year of age may evolve to a highly lethal course characterized by hypoxemia and refractory pulmonary hypertension, being this responsible for cardiovascular collapse. Pulmonary hypertension occurs by direct action of bacteria and its toxins, responsible for the vascular tone alteration associated with hyperviscosity syndrome and arteriolar thrombosis due to hyperleukocytosis. Since 2004, leucodepletion has been proposed as a treatment option; there are currently 18 publications on leucodepletion in patients with severe Pertussis, with a total of 288 patients, 68 cytoreduction procedures and a mortality rate of 28% in those exposed to the mentioned treatment. Published studies have design limitations, so the evidence for using leukoreduction in patients with severe pertussis remains low.