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 αIIbß3 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 region 2 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 in vivo 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 thromboxane A2 analog U46619. Shp2 appears to act downstream of integrin αIIb ß3 outside-in signaling, inhibiting the phosphorylation of Akt (Ser473 and Thr308) and dense granule secretion. Calmodulin was also shown to bind 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.

Endothelial Cell-Derived von Willebrand Factor Is the Major Determinant That Mediates von Willebrand Factor-Dependent Acute Ischemic Stroke by Promoting Postischemic Thrombo-Inflammation.

OBJECTIVE: von Willebrand factor (VWF), which is synthesized in endothelial cells and megakaryocytes, is known to worsen stroke outcome. In vitro studies suggest that platelet-derived VWF (Plt-VWF) is biochemically different from the endothelial cell-derived VWF (EC-VWF). However, little is known about relative contribution of different pools of VWF in stroke. APPROACH AND RESULTS: Using bone marrow transplantation, we generated chimeric Plt-VWF mice, Plt-VWF mice that lack ADAMTS13 in platelets and plasma (Plt-VWF/Adamts13(-/-)), and EC-VWF mice to determine relative contribution of different pools of VWF in stroke. In brain ischemia/reperfusion injury model, we found that infarct size and postischemic intracerebral thrombo-inflammation (fibrin(ogen) deposition, neutrophil infiltration, interleukin-1ß, and tumor necrosis factor-α levels) within lesions were comparable between EC-VWF and wild-type mice. Infarct size and postischemic thrombo-inflammation were comparable between Plt-VWF and Plt-VWF/Adamts13(-/-) mice, but decreased compared with EC-VWF and wild-type mice (P<0.05) and increased compared with Vwf(-/-) mice (P<0.05). Susceptibility to FeCl3 injury-induced carotid artery thrombosis was comparable between wild-type and EC-VWF mice, whereas Plt-VWF and Plt-VWF/Adamts13(-/-) mice exhibited defective thrombosis. Although most of the injured vessels did not occlude, slope over time showed that thrombus growth rate was increased in both Plt-VWF and Plt-VWF/Adamts13(-/-) mice compared with Vwf(-/-) mice (P<0.05), but decreased compared with wild-type or EC-VWF mice. CONCLUSIONS: Plt-VWF, either in presence or absence of ADAMTS13, partially contributes to VWF-dependent injury and postischemic thrombo-inflammation after stroke. EC-VWF is the major determinant that mediates VWF-dependent ischemic stroke by promoting postischemic thrombo-inflammation.

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury.

AIM: To characterize high-mobility group protein 1-toll-like receptor 4 (HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion (I/R) injury. METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups (n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88 (MyD88), and anti-translocating-chain-associating membrane protein (TRIF) antibody groups. Vehicle with the control IgG antibody, anti-HMGB1, anti-MyD88, or anti-TRIF antibodies (all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor (NF)-κB p65, interleukin (IL)-6, and tumor necrosis factor (TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. In addition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of mRNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance. RESULTS: Blocking HMGB1, MyD88, and TRIF expression by injecting anti-HMGB1, anti-MyD88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81 (P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38 (P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63 (P < 0.05) for the sham, control, anti-HMGB1, anti-MyD88, and anti-TRIF groups, respectively (all in pg/mL).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of anti-HMGB1, anti-MyD88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect. CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Defective thrombus formation in mice lacking endogenous factor VII activating protease (FSAP).

Factor VII (FVII) activating protease (FSAP) is a circulating protease with a putative function in blood coagulation and fibrinolysis. Genetic epidemiological studies have implied a role for FSAP in carotid stenosis, stroke and thrombosis. To date, no in vivo evidence is available to support these claims. We have, for the first time, used FSAP-/- mice to define its role in thrombosis and haemostasis in vivo and to characterise the molecular mechanisms involved. FeCl3-induced arterial thrombosis in carotid and mesenteric artery revealed that the occlusion time was significantly increased in FSAP-/- mice (p< 0.01) and that some FSAP-/- mice did not occlude at all. FSAP-/- mice were protected from lethal pulmonary thromboembolism induced by collagen/ epinephrine infusion (p< 0.01). Although no spontaneous bleeding was evident, in the tail bleeding assay a re-bleeding pattern was observed in FSAP-/- mice. To explain these observations at a mechanistic level we then determined how haemostasis factors and putative FSAP substrates were altered in FSAP-/- mice. Tissue factor pathway inhibitor (TFPI) levels were higher in FSAP-/- mice compared to WT mice whereas FVIIa levels were unchanged. Other coagulation factors as well as markers of platelet activation and function revealed no significant differences between WT and FSAP-/- mice. This phenotype of FSAP-/- mice could be reversed by application of exogenous FSAP. In conclusion, a lack of endogenous FSAP impaired the formation of stable, occlusive thrombi in mice. The underlying in vivo effect of FSAP is more likely to be related to the modulation of TFPI rather than FVIIa.

Role of calreticulin mutations in the aetiological diagnosis of splanchnic vein thrombosis.

BACKGROUND & AIMS: Myeloproliferative neoplasms are the most common aetiological cause of splanchnic vein thrombosis (SVT). In these patients, the JAK2V617F mutation has facilitated the diagnosis of an underlying myeloproliferative neoplasm (MPN). Recently, somatic mutations of the calreticulin (CALR) gene have been identified in MPN patients lacking the JAK2 mutation. The aim of the present study was to ascertain whether CALR mutations could also play a role in the diagnosis of masked MPN in SVT. METHODS: We included 209 patients with SVT (140 with PVT and 69 with Budd-Chiari syndrome) who had a complete aetiological diagnostic work-out. They were investigated for CALR mutations. RESULTS: CALR mutations were found in 4 of the 209 patients (1.9%). They represented 5.4% of patients with an underlying MPN of whom all had already been diagnosed with a MPN using conventional criteria including bone marrow biopsy findings. CONCLUSIONS: In the screening of underlying MPNs in patients with SVT, given its high frequency in these disorders, the JAK2 mutation must be evaluated first and, if negative, CALR mutations should also be investigated. This approach would increase the diagnostic yield of masked MPNs by reducing the need for additional studies.

Mesenteric vein thrombosis in a patient heterozygous for factor V Leiden and G20210A prothrombin genotypes.

Mesenteric venous thrombosis (MVT) is a rare but life threatening form of bowel ischemia. It is implicated in 6%-9% of all cases of acute mesenteric ischemia. The proportion of patients with primary (or idiopathic) MVT varies from 0% to 49%, with a decrease in frequency secondary to more recent availability of newer investigations for hypercoagulability. The presence of factor V Leiden (FVL) and prothrombin G20210A mutations (PGM) have been well documented in these cases. However, there have been scarce case reports describing MVT in heterozygotes of both these mutations occurring simultaneously and its implications on long term management. Our case describes acute MVT in a previously asymptomatic young patient with no prior history of venous thromboembolism. The patient was found to be heterozygous for FVL and PGM and treated with lifelong anticoagulation with warfarin (goal international normalized ratio: 2-3) and avoidance of hormonal contraceptives.

Preconditioning with soluble guanylate cyclase activation prevents postischemic inflammation and reduces nitrate tolerance in heme oxygenase-1 knockout mice.

Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²âº. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function.

Prothrombotic disorders in abdominal vein thrombosis.

Abdominal vein thrombosis is a rare, but potentially life-threatening form of venous thrombosis. It mainly involves the hepatic veins (Budd Chiari syndrome, BCS), portal veins (PVT) and mesenteric veins. In recent years several large-scale studies have been performed to study the underlying aetiological factors in these thrombotic disorders. Both inherited and acquired thrombophilia factors are frequently observed in these patients. Factor V Leiden mutation is frequently found in patients with BCS and prothrombin gene variant is seen more frequently in PVT. Myeloproliferative neoplasms (MPNs), including polycythemia vera and essential thrombocythemia, are underlying disorders in 30-40% of patients with abdominal vein thrombosis. Other aetiological factors are paroxysmal nocturnal haemoglobinuria (PNH), autoimmune disorders and hormonal factors. Recently, several new risk factors have been reported and are discussed in this review. BCS and PVT are multi-factorial disorders. In nearly 50% of patients two, and in 16% even three prothrombotic risk factors were found at presentation. Because patients with abdominal vein thrombosis have a high risk of recurrence immediate anticoagulant treatment is necessary. The duration of treatment is still a matter of debate because these patients also have a high risk of bleeding, especially those with portal hypertension. For BCS patients life-long anticoagulant treatment is advised. In patients with PVT it is recommended to tailor treatment to the individual patient based on the presence of an underlying prothrombotic disorder and the risk of bleeding.

Essential domains of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 metalloprotease required for modulation of arterial thrombosis.

OBJECTIVE: A disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) inhibits platelet aggregation and arterial thrombosis by cleavage of von Willebrand factor. However, the structural components of ADAMTS13 required for inhibition of arterial thrombosis are not fully defined. METHODS AND RESULTS: Using recombinant proteins and a murine model, we demonstrated that an ADAMTS13 variant truncated after either the eighth thrombospondin type 1 repeat or the spacer domain inhibits ferric chloride-induced arterial thrombosis in ADAMTS13(-/-) mice with efficacy similar to that of full-length ADAMTS13. The results obtained from monitoring thrombus formation in carotid and mesenteric arteries were highly concordant. Further analyses by site-directed mutagenesis and human monoclonal antibody inhibition assay revealed that the Cys-rich and spacer domains of ADAMTS13, particularly the amino acid residues between Arg559 and Glu664 in the spacer domain, may be critical for modulation of arterial thrombosis in vivo. Finally, the thrombosis-modulating function of ADAMTS13 and variants/mutants was highly correlated with the von Willebrand factor-cleavage activity under fluid shear stress. CONCLUSIONS: Our results suggest that the amino terminus of ADAMTS13, specifically the variable region of the spacer domain, is crucial for modulation of arterial thromboses under (patho)physiological conditions. These findings shed more light on the structure-function relationship of ADAMTS13 in vivo and may be applicable for rational design of protein- or gene-based therapy of arterial thromboses.

Mesenteric venous thrombosis in a child with type 2 protein S deficiency.

A 5-year-old girl presented with abdominal pain and bloody stools 2 weeks after suffering from influenza A infection. Enhanced computed tomographic scan showed widespread splanchnic venous thrombosis and small intestine necrosis. She recovered after the necrotic bowel was resected. The patient continues to receive anticoagulant therapy. Thrombophilia screening after the complete resolution consistently showed mildly decreased protein S (PS) activity with normal PS antigen levels. Sequence analysis detected a heterozygous K196E mutation in the PROS1 gene. Type 2 PS deficiency was diagnosed. This is the first report of mesenteric vein thrombosis in a child with a type 2 PS deficiency.

Altered mRNA expression due to acute mesenteric ischaemia in a porcine model.

INTRODUCTION: Messenger RNA (mRNA) changes in the small intestine in response to acute mesenteric ischaemia (AMI) could offer novel diagnostic possibilities, but have not been described. The aim was to characterize the mRNA response to experimental AMI. MATERIALS AND METHODS: Twelve pigs underwent catheterisation of the superior mesenteric artery with injection of polivinylalcohol embolisation particles or sodium chloride. Laparotomy and intestinal tissue sampling were performed. Microarray analysis was performed using the GeneChip(®) whole porcine genome array. RESULTS: Seven down-regulated cellular pathways were associated with protein, lipid and carbohydrate metabolism. Seventeen up-regulated pathways were associated with inflammatory and immunological activity, regulation of extracellular matrix and decreased cellular proliferation. Thrombospondin (THS), monocyte chemoattractant protein 1(MCP-1) and gap junction alpha 1(GJA-1) were consistently up-regulated in all embolised pigs. Genes encoding earlier proposed biomarkers for AMI were up-regulated, such as lactate dehydrogenase and creatine kinase, or down-regulated, such as intestinal fatty acid binding protein and glutathione S-transferase. CONCLUSION: This study describes the intestinal tissue response on a gene expression level to AMI. THS, MCP-1 and GJA-1 were consistently up-regulated by ischaemia, whereas earlier proposed biomarkers for AMI were not. Gene expression may not be directly linked to the use of the corresponding proteins as potential clinical biomarkers.

JAK2 V617F mutation, mesenteric vein thrombosis, and myeloproliferative disorders.

Mesenteric vein thrombosis is a rare disorder that is often the first manifestation of a systemic condition such as a hypercoagulable state or cancer. In particular, myeloproliferative disorders can present as mesenteric vein thrombosis even in the setting of relatively normal peripheral blood counts. A recent novel mutation in the Janus activated kinase 2 gene involving a gain-of-function substitute of valine to phenylalanine at position 617 (JAK2 V617F) has been discovered to be prevalent in patients with mesenteric vein thrombosis and myeloproliferative disorders. This article reports a patient who presented with mesenteric vein thrombosis and relatively normal peripheral blood counts. He was diagnosed with essential thrombocythemia after he tested positive for the JAK2 V617F mutation.

Hyperhomocysteinaemia, low folate concentrations and methylene tetrahydrofolate reductase C677T mutation in acute mesenteric venous thrombosis.

OBJECTIVES: Acute mesenteric venous thrombosis (AMVT) was first reported by Fagge and was recognised as a distinct clinical entity by Warreen in 1935. However, its pathogenesis is still unclear. Elevated plasma levels of homocysteine (Hcy) are associated with an increased risk of deep vein thrombosis. This case-control study examines the potential association among hyperhomocysteinaemia (hyper-Hcy), low serum folate and vitamin B(12) levels and the common C677T mutation of the MTHFR gene in patients with AMVT. MATERIALS AND METHODS: Sixty-three patients with AMVT and 75 sex- and age-matched healthy controls were recruited, and their plasma Hcy, folate and vitamin B(12) levels were measured by high performance liquid chromatography (HPLC) and immunological assays. The polymorphism of MTHFR C677T was detected by PCR-RFLP. RESULTS: The mean plasma Hcy levels were significantly higher in patients with AVMT compared with controls (23.5 standard deviation (S.D.) 8.8 vs. 12.6+/-6.6micromoll(-1), P<0.01). The fasting Hcy correlated negatively with folate (AMVT: r=-0.42, P<0.01; CONTROL: r=-0.40, P<0.01). The frequency of homozygous (TT) genotype in MTHFR C677T mutation was significantly higher in patients with AMVT than that in control subjects (33% vs. 17%; chi square (chi(2))=6.31, P<0.05; odds ratio (OR)=2.80; 95% confidence interval (CI): 1.25-6.25). Compared with the control subjects, the mean serum vitamin B(12) levels were lower in patients, but it was not statistically significant (365+/-88pmoll(-1) vs. 408+/-108pmoll(-1), P>0.05). CONCLUSIONS: Hyper-Hcy and low serum folate levels were associated with an increased risk of AMVT. The homozygous (TT) genotype of MTHFR gene mutation may be a crucial hereditary risk factor in the development of AMVT for a Chinese population.

Mesenteric venous thrombosis with bowel infarction and hyperhomocysteinemia due to homozygous methylenetetrahydrofolate reductase C677T genotype.

The case of a 30-year-old man with bowel infarction due to mesenteric venous thrombosis and multiple risk factors, including mild hyperhomocysteinemia due to methylenetetrahydrofolate reductase C677T polymorphism and recent abdominal surgery, is reported. His clinical manifestation consisted of persistent abdominal pain; complementary examinations showed nonspecific findings such as leukocytosis and dilated loops of the bowel. The diagnosis of mesenteric venous thrombosis with bowel infarction was made during laparotomy and confirmed by anatomopathologic examination. He underwent segmental resection associated with lifelong anticoagulant therapy and vitamin B supplementation with a favorable course.

[APC resistance due to factor V Leiden mutation presenting as acute hemorrhagic jejunal infarction--case report]. / Akuter hämorrhagischer Jejunuminfarkt als Erstmanifestation einer APC-Resistenz bei Faktor-V-Leiden-Mutation--ein Fallbericht.

We report on a 43-year-old patient presenting to the emergency department with acute abdominal pain the source of which turned out to be acute hemorrhagic jejunal infarction due to portal and mesenteric vein occlusion with no apparent cause. In spite of a lacking history of hereditary thrombophilic risk factors, further diagnostic procedures revealed heterozygous factor V Leiden mutation. Diagnosis, therapy and clinical course are described. An overview on acute mesenteric venous occlusion with special reference to genetically determined thrombophilic disorders is given.

The JAK2 V617F mutation frequently occurs in patients with portal and mesenteric venous thrombosis.

BACKGROUND: Myeloproliferative disorders (MPDs) represent a risk factor for thrombosis in the portal, mesenteric, and hepatic districts. OBJECTIVE: We aimed to assess whether the Janus kinase 2 (JAK2) V617F mutation, an acquired mutation that occurs in MPD patients, is a risk factor for portal and mesenteric venous thrombosis (PMVT) independently of the presence of overt MPDs. PATIENTS AND METHODS: The medical histories of 99 patients presenting with PMVT were obtained. The presence of the JAK2 V617F and VHL 598C > T mutations was determined by polymerase chain reaction followed by restriction enzyme analysis and direct cycle sequence analysis. RESULTS: Over a 10-year period of observation, of the 99 patients presenting with PMVT, the JAK2 V617F mutation was detected in heterozygous state in 17 individuals [17.2%; 95% confidence interval (95% CI) 10.9-25.9]. None of the patients presenting with the JAK2 V617F mutation carried an inherited thrombophilic risk factor. Seven patients with (43.8%; 95% CI 19.8-70.1) and two without (2.4%; 95% CI 0.3-8.4) the JAK2 V617F mutation had a diagnosis of MPD at the occurrence of the venous thrombotic event. After a median follow-up of 41 months (range 3-114 months), three out of the 10 patients carrying the JAK2 V617F mutation were then diagnosed as having idiopathic myelofibrosis (n = 2) or polycythemia vera (n = 1), whereas in seven patients a MPD was not detected. Two of the 83 patients without the JAK2 V617F mutation went on to develop MPDs. CONCLUSIONS: Determination of the JAK2 V617F mutation may contribute to the search for genetic determinants of PMVT and may be useful to recognize patients who should be carefully observed for the subsequent development of overt MPDs.

Heterozygous prothrombin 20210G/A mutation, associated with hyperhomocysteinemia, and homozygous methylenetetrahydrofolate reductase 677C/T mutation, in a patient with portal and mesenteric venous thrombosis.

We herein report a 34-year-old man who was investigated for severe abdominal pain. Portal vein thrombosis (PVT) and mesenteric vein thrombosis (MVT) were diagnosed. An association with two predisposing factors for thrombosis was noted: (1) heterozygous factor II 20210G/A mutation and (2) homozygous methylenetetrahydrofolate reductase (MTHFR) 677C/T mutation with hyperhomocysteinemia. Our case is of particular interest because the patient reported herein, is homozygote for the MTHFR 677C/T mutation, while the only two other cases reported in the literature with similar gene mutations, were heterozygotes for the mutation.