bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Mutations in the Canine von Willebrand Factor Gene Associated with Type III von Willebrand Disease
Authors: Rieger M. , Schwarz H. P. , Turecek P. L. , Dorner F. , Mourik van , Mannhalter C.
Published: March 2018

In humans, type III von Willebrand disease is caused by deletions or nonsense mutations. In dogs, the underlying genetic defects have not been determined yet. We searched for the genetic defect in four related type III deficient Dutch Kooiker dogs obtained from one breeder. Mutation analysis was performed with total RNA isolated from platelets or whole blood. The complete coding region of the vWf gene was amplified by RT-PCR and sequenced by the cycle sequencing technique. Two homozygous mutations were found, a G→A transition at the first position of the donor splice site sequence of intron 16 (TGgtaagt→TGataagt) and a missense mutation at nt 208 (G→A) (1). The splice site defect resulted in the generation of a transcript containing 46bp of intron sequence and a stop codon at amino acid position 729 in the propeptide region of the vWf protein. This mutation seems to be causative for the type III phenotype. The effect of the missense mutation in exon 3 which causes a change of Val to Ile on the vWD phenotype is unclear. Probably, this transition represents a polymorphism occurring in Dutch Kooiker dogs. Both mutations were not present in 5 healthy mongrel dogs.

Parts of this paper were presented at the 39th annual meeting of the American Society of Hematology (ASH), December 5-9, San Diego, USA
The Molecular Basis of Antithrombin Deficiency in Belgian and Dutch Families
Authors: Lissens W. , Seneca S. , Capel P. , Chatelain B. , Meeus P. , Osselaer J. C. , Peerlinck K. , Seghers J. , Slacmeulder M. , Stibbe J. , Loo J. van de , Vermylen J. , Liebaers I. , De Waele M. , Jochmans K.
Published: March 2018

The molecular basis of hereditary antithrombin (AT) deficiency has been investigated in ten Belgian and three Dutch unrelated kindreds. Eleven of these families had a quantitative or type I AT deficiency, with a history of major venous thromboembolic events in different affected members. In the other two families a qualitative or type II AT deficiency was occasionally diagnosed.

DNA studies of the AT gene were performed, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and intronexon junction regions. Six novel point mutations were identified: four missense, one nonsense mutation and a single nucleotide deletion near the reactive site, causing a frameshift with premature translation termination. In two kindreds the underlying genetic defect was caused by a whole gene deletion, known as a rare cause of AT deficiency. In these cases, Southern blot and polymorphism analysis of different parts of the AT gene proved useful for diagnosis. In another kindred a partial gene deletion spanning 698 basepairs could precisely be determined to a part of intron 3B and exon 4. In two type I and in both type II AT deficient families a previously reported mutation was identified. In all cases, the affected individuals were heterozygous for the genetic defect.
Effect of Cilostazol on Soluble Adhesion Molecules and Platelet-derived Microparticles in Patients with Diabetes
Authors: Shouzu Akira , Omoto Seitaro , Hayakawa Takashi , Kagawa Hideo , Nishikawa Mitsushige , Inada Mitsuo , Fujimura Yoshihiro , Ikeda Yasuo , Fukuhara Shirou , Nomura Shosaku
Published: March 2018

We evaluated the plasma concentrations of soluble adhesion molecules and platelet-derived microparticles (PMP) in patients with non-insulin dependent diabetes mellitus (NIDDM) and studied the effect of cilostazol on PMP generation. There were differences in the levels of soluble adhesion molecules between NIDDM patients (N = 43) and the control subjects (N = 30) (soluble thrombomodulin: 11.5 ± 5.3 vs. 7.0 ± 1.2 TU/ml, p<0.0001; soluble vascular cell adhesion molecule-1: 708 ± 203 vs. 492 ± 113 ng/dl, p<0.0001; soluble intercellular cell adhesion molecules-1: 274 ± 65 vs. 206 ± 48 ng/dl, p<0.0001; soluble P-selectin: 194 ± 85 vs. 125 ± 43 ng/dl, p<0.0001). There were also differences in the levels of PMP and platelet activation markers between NIDDM patients and the controls (PMP: 943 ± 504 vs. 488 ± 219/10 4 plt, p<0.0001; platelet CD62P: 9.2 ± 4.6 vs. 4.4 ± 4.3%, p<0.001; platelet CD63: 10.2 ± 4.5 vs. 4.5 ± 3.3%, p<0.0001; platelet annexin V: 9.1 ± 3.9 vs. 5.3 ± 3.8%, p<0.001). To study the release of PMP into plasma, a modified cone-and-plate viscometer was used. Increased release of PMP from platelets was observed in diabetic plasma compared to normal plasma under high shear stress conditions (2,672 ± 645 vs. 1,498 ± 386/104 plt, p<0.05). Therefore, one cause of PMP elevation in NIDDM may be high shear stress. The levels of PMP, activated platelets, and soluble adhesion molecules all decreased significantly after treatment with cilostazol. These results suggest that cilostazol may be useful for the inhibition of both PMP-dependent and -independent vascular damage in NIDDM.
Preinfarction Angina and Improved Reperfusion of the Infarct-related Artery
Authors: Sciahbasi Alessandro , De Marco Eugenia , Maseri Attilio , Andreotti Felicita
Published: March 2018

Preinfarction angina and early reperfusion of the infarct-related artery are major determinants of reduced infarct-size in patients with acute myocardial infarction. The beneficial effects of preinfarction angina on infarct size have been attributed to the development of collateral vessels and/or to post-ischemic myocardial protection. However, recently, a relation has been found between prodromal angina, faster coronary recanalization, and smaller infarcts in patients treated with rt-PA: those with preinfarction angina showed earlier reperfusion (p = 0.006) and a 50% reduction of CKMB-estimated infarct-size (p = 0.009) compared to patients without preinfarction angina. This intriguing observation is consistent with a subsequent observation of higher coronary recanalization rates following thrombolysis in patients with prodromal preinfarction angina compared to patients without antecedent angina. Recent findings in dogs show an enhanced spontaneous lysis of plateletrich coronary thrombi with ischemic preconditioning, which is prevented by adenosine blockade, suggesting an antithrom-botic effect of ischemic metabolites. Understanding the mechanisms responsible for earlier and enhanced coronary recanalization in patients with preinfarction angina may open the way to new reperfusion strategies.

A vast number of studies, globally involving ≈17,000 patients with acute myocardial infarction, have unequivocally shown that an infarction preceded by angina evolves into a smaller area of necrosis compared to an infarct not preceded by angina (Table 1) (1). So far, preinfarction angina has been thought to have cardioprotective effects mainly through two mechanisms: collateral perfusion of the infarctzone (2-4), and ischemic preconditioning of the myocardium (5-7). Here we discuss a further mechanism of protection represented by improved reperfusion of the infarct-related artery.
Thrombolysis in Newborns and Infants
Authors: Auberger K. , Halimeh S. , Junker R. , Klinge J. , Kreuz W. D. , Ries M. , Schlegel N. , Nowak-Göttl U.
Published: March 2018

This review analyses literature reports from 1970 to 1998 assessing the use of streptokinase (SK), urokinase (UK) or recombinant tissuetype plasminogen activator (rt-PA) for thrombolytic therapy in neonates and infants. From 1970 to 1998 182 infants were reported to have received SK (n = 54; 29.5%), UK (n = 41; 22.5%) or rt-PA (n = 87; 48%). During thrombolytic therapy no concomitant heparin administration or low dose heparin therapy (5 U/kg/h) were recorded. To perform reocclusion prophylactics heparin was reinitiated at the end of thrombolytic therapy usually in the recommended dosage of 20 U/kg/h. The overall thrombolytic patency rate in neonates varied from 39% to 86%. Besides bleeding from local puncture sites or recent catheterisation sites (10.4%), pulmonary embolism was reported in 1.1% of the 182 infants. Major bleeding complications, i.e. pulmonary bleeding (0.6%), gastrointestinal bleeding (0.6%) or intraventricular haemorrhage (IVH 2.7%) are rarely reported side effects and only 2 thrombolysis related deaths due to haemorrhage were mentioned. Bleedings reported in the central nervous system (n = 4) mainly occurred in preterm infants (n = 3). In conclusion, data of this preliminary analysis suggest that there is no big difference (p = 0.09; χ2-test) in the efficacy rate between the 3 thrombolytic agents used in the first year of life. In each case an assessment must be made with respect to the relative benefit conferred by thrombolytic therapy in preventing organ or limb damage versus the potential side effects, costs and inconvenience for the childhood patient. Controlled prospective multicentre studies on thrombolytic therapy in neonates and infants are recommended to evaluate patency rates and adverse effects for the different thrombolytic agents used.
Ultrasound Thrombolysis
Authors: Gaul Georg B.
Published: March 2018

The possibility of lysing clots by the direct intravascular application of ultrasound was described first in 1974 in an animal experiment. However research on therapeutic ultrasound for thrombolysis gained momentum in the early 1980s and is now divided into several directions: 1) pharmacological thrombolysis supported by externally applied ultrasound; 2) pharmacological thrombolysis accelerated by intravascular ultrasound; 3) lysis of intravascular clots by intravascular ultrasound applied as singular treatment; 4) facilitating angioplasty by intravascular ultrasound which may smoothen the rigid surface of calcified arteries by lysing calcified structures out of sclerotic plaques.

In acute myocardial infarction first evidence of efficacy in lysing clots by ultrasound was described in two small series of 15 patients in 1997. Furthermore, ultrasound thrombolysis seems to be efficient also in occluded saphenous vein grafts and in completely obstructed peripheral arteries, where the latest clinical experience gives very promising results.
Depletion of Plasma Factor XIII Prevents Disseminated Intravascular Coagulation-induced Organ Damage
Authors: Lee Soo Young , Chang Sung Keun , Lee Il Ha , Kim Yu-Mie , Chung Soo Il
Published: March 2018

The impact of clot stability affecting the vasculopathy and tissue necrosis in Shwartzman reaction was investigated using plasma Factor XIII A2-depleted rabbit (FXIII-DR). Plasma Factor XIIIA2 (FXIIIA2) was depleted by infusion of the mono-specific goat anti-rabbit FXIIIA2 IgG. Generalized Shwartzman reaction (GSR) was induced by priming and challenged by i.v. injection of LPS and local Shwartzman reaction (LSR) was primed by intradermal injection of LPS and challenged by i.v. injection of LPS. Histological examination of the GSR animals showed, extensive thrombi accumulation in renal tubules and bilateral cortical necrosis of kidney in 8 out of 10 rabbits but none in the FXIIIDR. Fibrinogen levels were elevated to 3~4 fold at 24 h and lowered at 48 h whereas a steady rise was seen in the FXIII-DR. FDP levels in GSR animals were significantly elevated at 24 h and further increased at 48 h but only slightly elevated in the FXIII-DR. Examination of the LSR tissues after 48 h showed an acute onset of progressive cutaneous vascular thrombosis, purpura, and secondary hemorrhagic necrosis whereas neither fibrin deposit nor necrosis of tissue were detected in FXIII-DR despite of an early edema formation. Fibrinogen levels were also increased two fold at 24 h but returned to basal levels at 48 h in control LSR animals but not affected at all in FXIII-DR. These results suggest that during the severe inflammatory conditions such as sepsis, the fibrinolytic system is functionally sufficient to dissipate the pathogenic accumulation of disseminated intravascular clots and exudated fibrin clots if those clots were prevented from getting cross-linked in plasma.
Inhibition of Arterial Thrombosis by a Soluble Tissue Factor Mutant and Active Site-blocked Factors IXa and Xa in the Guinea Pig
Authors: Refino Canio , Burcklen Louis , Himber Jacques , Roux Sébastien , Kirchhofer Daniel
Published: March 2018

The substrate recognition region of tissue factor contains two residues, Lys165 and Lys166, which are important for macromolecular substrate activation by the tissue factor:factor VIIa complex. Replacement of these two residues with alanine in a soluble version of human tissue factor resulted in a mutant, hTFAA, which can bind factor VIIa but forms an enzymatically inactive complex. We found that hTFAA inhibits the activity of guinea pig factor VIIa, allowing us to evaluate hTFAAs effects on thrombosis and hemostasis in a guinea pig model of recurrent arterial thrombosis. In addition to heparin, the effects of hTFAA were compared to active site inhibited factor IXa (F.IXai) and factor Xa (F.Xai). We found that hTFAA, F.IXai and F.Xai were potent antithrombotics and may possess a decreased risk of hemorrhage when compared to unfractionated heparin. When administered at a dose that inhibited thrombosis by about 90%, hTFAA neither affected cuticle bleeding nor the activated partial thromboplastin time, and had only a modest effect on the prothrombin time. At equi-efficacious doses, F.IXai, F.Xai and heparin prolonged bleeding times by 20% (p >0.5), 50% (p <0.05) and 100% (p <0.01), respectively. In summary, our study demonstrates that, unlike heparin, specific inhibitors of factors VIIa, IXa and Xa can produce antithrombotic effects without or with only minimally disturbing normal hemostasis. The results further suggest that factor VIIa and factor IXa are especially promising targets for antithrombotic drug development.
Contribution of Platelet-derived Factor Va to Thrombin Generation on Immobilized Collagen- and Fibrinogen-adherent Platelets
Authors: Briedé Jacob , Heemskerk Johan , Cornelis van’t Veer , Hemker H. Coenraad , Lindhout Theo
Published: March 2018

Adhesion of platelets to immobilized collagen induces the expression of anionic phospholipids, e. g. phosphatidylserine (PS), in the outer leaflet of the plasma membrane of these platelets. In contrast, of the platelets that adhere to immobilized fibrinogen only a small sub-population representing 10 ± 3% of the total population of the fibrinogen-adherent platelets has exposed PS as probed by annexin V binding. Although the presence of PS is thought to be critical for thrombin generation at the platelet surface, no information is available about the effect of this differential PS exposure on the ability of adherent platelets to support thrombin generation. Perfusion of the fibrinogen- or collagen-adherent platelets with solutions containing factor Xa and prothrombin resulted in thrombin generation that i) increased linear during the first perfusion minutes, ii) was about two-fold faster at collagen-adherent than at fibrinogen-adherent platelets and iii) was for more than 98% restricted to the surface of the adherent platelets. It appeared that the lower thrombin generating capacity of fibrinogen-adherent platelets is not due to a lower overall surface density of PS, but is caused by lower amounts of platelet-bound factor Va. Firstly, in both cases thrombin generation could be completely attenuated with antibodies against human factor Va, and secondly, in the presence of an excess of exogenous plasma-derived factor Va similar initial rates of thrombin formation were measured for collagen- and fibrinogen-adherent platelets. Our findings suggest a unique role for immobilized collagen in maintaining haemostasis.
Thrombopoietin: From Theory to Reality
Authors: Kaushansky Kenneth
Published: March 2018

Introduction

The term thrombopoietin (TPO) was first penned in 1958 to describe the primary regulator of platelet production. Multiple efforts to purify the hormone were unsuccessful throughout the 1970s and 1980s because the protein was scarce and starting materials for purification biochemically complex. Even purification of thrombocytopenic plasma, which is the richest source of TPO, was unsuccessful.1,2 However, the cloning of the proto-oncogene c-mpl
3 opened an alternative route to the identification of TPO. Detailed study of the c-mpl gene revealed that it encoded an orphan hematopoietic cytokine receptor. Its tissue distribution and experiments designed to eliminate its expression suggested that c-mpl encoded the TPO receptor.4,5 In 1994, using three distinct strategies, five separate groups either purified protein or cloned cDNA for TPO.6 This review will focus on the physiology of TPO production and function, concentrating on recent findings that help to explain platelet homeostasis and how our understanding of TPO can translate into better patient care for thrombocytopenic patients.
The Elegant Platelet: Signals Controlling Actin Assembly
Authors: Barkalow Kurt , Azim Anser , Italiano Joe , Hartwig John
Published: March 2018

The function of the blood platelet is defined by its two physical states. The platelet is born in the first state, in long cytoplasmic extensions from megakaryocytes, as a small disc of reproducible structure having an elegant, actin-based cytoskeleton. At rest, the platelet circulates through the vasculature in this form. In the second state, in response to vascular damage, the platelet rapidly converts into its active form with filopodia and lamellipodia that derive from a remodeled actin skeleton and a massive assembly of new actin filaments. In the last two decades, we have begun to understand the structural basis for these two platelet states, the role of specific proteins that define the architecture of both shapes, and the proteins and signals that drive this conversion of shapes.
Molecular Risk Factors for Thrombosis
Authors: Bertina Rogier
Published: March 2018

Introduction

A diagnosis of venous thrombosis is made each year in about 0.1% of Western populations. In the majority of cases, the diagnosis is based on objective evidence for an obstruction of the circulation by a blood clot that either formed locally or developed elsewhere in the circulation. The interest in the pathogenesis of this hemostatic process has increased enormously during the past 20 years and, as a consequence, substantial progress has been made in understanding the multifactorial nature of the disease, where interactions between genetic and environmental factors result in the formation of an obstructive thrombus at a specific time and at a specific location. Frequently, these thrombi are found in the elderly in the superficial and deep veins of the legs. Thrombi may also occur in younger individuals and in the veins of the brain, mesentery, liver, or retina.

The episodic nature of the disease points out the importance of acquired and/or environmental factors in thrombosis. The disease also has strong genetic components, given the many reports of familial clustering of thrombotic events1-3 and the fact that 20% to 30% of patients with a first thrombotic event report at least one first-degree relative with thrombosis.4

The search for molecular risk factors for venous thrombosis remains intensive. Over the years, this research has been facilitated by new insights in the regulation of blood coagulation and by the development of new research methods, especially in the field of molecular genetics. Today, at least one genetic defect can be found in about 70% of the families with thrombophilia.5

Initial studies focussed on patients and their relatives (family studies). Such studies are very useful but can be hampered by selection bias. In other words, we know these families because the proband and some of his/her relatives have experienced thrombosis. Therefore, later studies used groups of unrelated patients and healthy controls (patient-control studies). One of the limitations of these studies is that they need to be very large to identify the less common risk factors.

The concept of venous thrombosis as a multifactorial disease has received much attention in recent years.6-8 One of the reasons is that some of the newly discovered genetic risk factors concern single point mutations that are quite common in the general population (e.g., factor V Leiden, prothrombin 20210A allele).9,10 As a result, studies focussing on the interaction of these mutations with other relatively common risk factors (e.g., use of oral contraceptives,11,12 pregnancy,13 surgery,14 but also lupus anticoagulant15 or mild hyperhomocysteinemia16,17) have become feasible. In addition, researchers will continue to learn more about the relationship between these common molecular risk factors and the clinical phenotypes, namely deep vein thrombosis, pulmonary embolism, superficial thrombosis, and first versus recurrent events.
Thromboembolism and Pregnancy
Authors: Ginsberg Jeffrey
Published: March 2018

Introduction

Antithrombotic therapy during pregnancy is used for the treatment and prophylaxis of venous thromboembolic disease, for the prevention and treatment of arterial embolism associated with valvular heart disease and/or prosthetic heart valves, for the prevention of fetal growth retardation and pregnancy loss in patients with antiphospholipid antibodies, and in patients with pregnancy-induced hypertension. Since antithrombotic agents have the potential to produce complications in both the mother and fetus, their use during pregnancy raises concerns. Guidelines are difficult to establish because the evidence upon which recommendations are based is derived primarily from poor-quality studies. This chapter reviews the fetal and maternal effects of antithrombotic agents including oral anticoagulants, unfractionated heparin, low molecular weight heparins (LMWH), and aspirin, and provides recommendations for their use during pregnancy.
Inherited Thrombophilia and Pregnancy Loss
Authors: Brenner Benjamin
Published: March 2018

Introduction

A successful pregnancy is dependent on the development of adequate placental circulation. Abnormalities of placental vasculature may result in a number of gestational pathologies, including first and second trimester miscarriages, intrauterine growth retardation (IUGR), intrauterine fetal death (IUFD), placental abruption, and preeclampsia.1

Approximately 5% of women experience two or more consecutive abortions. Habitual abortions, defined as three or more spontaneous recurrent pregnancy losses, may affect as many as 1% to 2% of women of reproductive age. The discovery of an association between recurrent pregnancy loss, and antiphospholipid antibodies, specifically lupus anticoagulant, and anticardiolipin antibodies increased interest in a possible acquired thrombotic autoimmune cause.

The inherited thrombophilias are a group of genetic disorders of blood coagulation resulting in an increased risk of thrombosis. Today, a full understanding of the inherited thrombophilias is becoming increasingly important in the management of high-risk gestations. Several reports over the last three years have suggested that not only are these disorders associated with an increased risk of thromboembolic disease during pregnancy and puerperium, but they are also associated with an increased incidence of vascular pathologies, resulting in poor gestational outcome.2

This review will cover recent data concerning thrombophilia and vascular placental pathology, potential pathophysiologic mechanisms for this association, and available therapeutic modalities for prevention of placental vascular thrombosis in order to maximize successful gestational outcome.
Long-Term Treatment of Venous Thromboembolic Disease
Authors: Hutten Barbara , Koopman Maria , Büller Harry , Prins Martin
Published: March 2018

Introduction

After an initial 5- to 10-day course of treatment with either unfractionated or low molecular weight heparin, secondary prophylaxis is usually prescribed to patients with venous thromboembolic disease for a variable period of time. The primary aim of both initial and secondary treatment is to prevent recurrent thrombotic events, including deep vein thrombosis and fatal pulmonary embolism. This objective has to be achieved at a minimal risk of bleeding. There is general consensus that the initial treatment should be with low molecular weight or unfractionated heparin. For secondary prophylaxis, however, there are currently several options. Among these are continued unfractionated heparin or low molecular weight heparin and oral vitamin K-antagonists. In addition, compression stockings and caval vein filters are available.

In this chapter, we will first examine the available evidence for each of these options, then discuss the results of randomized trials that have studied the duration of secondary prophylaxis. Finally, we will attempt to synthesize this information using decision analytic techniques.
Mechanisms of Hypercoagulability
Authors: Dahl Ola
Published: March 2018

Introduction

Patients undergoing major traumas, major joint replacement surgery, and neurosurgery are at high risk for postoperative deep vein thrombosis (DVT). Even if modern low molecular weight heparin is given as prophylaxis, the frequency of DVT remains high in these patients.1-7 This may indicate that some patients possess a certain “heparin resistance,” due probably due to an overwhelming procoagulant activity and/or because the hypercoagulable state lasts for a longer time than presently assumed. Tissue factor (TF) is the primary cellular initiator of blood coagulation and the most potent trigger of blood clotting known today.8 Adventitia surrounding the major blood vessels, the brain, and the bone marrow are structures with high levels of TF.9 Elective hip replacement surgery, which is a fairly well-standardized procedure that involves a great deal of damage to the bone marrow, is therefore, well-suited as a model to study the mechanisms of hypercoagulability.
Anticoagulation in the Setting of Epidural or Spinal Anesthesia/Analgesia
Authors: Thompson Gale
Published: March 2018

Introduction

The focus of this chapter is on the word “analgesia,” as used in the title. Although the traditional role of the anesthesiologist is to provide anesthesia, or total pain relief, for surgical procedures in the operating room, it is no longer possible to be considered a “compleat” anesthesiologist by ending patient care at the time that the surgical patient is turned over to the recovery room nurse. There is a growing specialty and societal expectation that the anesthesiologist will smooth the pathway for recovery by using specific drugs and techniques to control postoperative pain. Usually the postoperative period includes a period of 1 to 5 days.

Why has the treatment of postoperative pain become such an issue? Can postoperative pain be effectively controlled in a more cost-effective manner than through the use of parenteral narcotics? In reality, narcotic drugs are not a cheap or effective approach. For example, any patient or nurse who has experienced post-thoracotomy pain control via the epidural route vs parenteral injections will testify to a preference for the epidural.1,2 When pain treatment is effective, there are multiple physiologic and economic spin-offs, such as better wound healing, decreased pulmonary infection rates, improved gastrointestinal function, shortened hospitalization time, and considerable savings of health care dollars.3-7
Reduction of Pain Episodes and Prothrombotic Activity in Sickle Cell Disease by Dietary n-3 Fatty Acids
Authors: Tomer Aaron , Kasey Suha , Connor William , Clark Scott , Harker Laurence , Eckman James
Published: March 2018

The effects of dietary n-3 fatty acids (n-3FAs) on the frequency of pain episodes and ex vivo blood tests of thrombosis have been evaluated in patients with sickle cell disease (SCD) utilizing a doubleblind, olive oil-controlled clinical trial. Dietary n-3FA therapy (0.1 g/kg/d) was provided as menhaden fish oil (0.25 g/kg/d) containing 12% eicosapentaenoic acid (EPA), and 18% docosahexaenoic acid (DHA). Within 1 month dietary n-3FAs exchanged with n-6FAs in plasma and erythrocyte membrane phospholipids (p <0.01 in all cases).

Treatment with dietary n-3FAs for 1 year reduced the frequency of pain episodes requiring presentation to the hospital from 7.8 events during the preceding year to 3.8 events/year (p <0.01; n = 5). By contrast, subjects receiving control dietary olive oil (n = 5) experienced 7.1 pain events/year, compared to 7.6 during the previous year (p >0.4). The reduction in episodes in n-3FA-treated subjects was also significant when compared to control subjects (p <0.01). Dietary n-3FA therapy was not associated with hemorrhagic, gastrointestinal or other adverse effects.

Compared to 10 asymptomatic African-American controls, sickle cell subjects demonstrated significantly increased pretreatment: 1) flow cytometric expression of platelet membrane P-selectin (CD62p; p <0.01) and annexin V binding sites (p = 0.02); 2) plasma levels of platelet-specific secretory proteins platelet factor 4 (PF4) and -thromboglobulin (TG) (p <0.01 in both cases); 3) plasma products of thrombin generation, prothrombin fragment 1.2 (F1.2) and thrombin:antithrombin (TAT) complex (p <0.01 in both cases); and 4) plasma levels of thrombolytic products, D-dimer and plasmin:antiplasmin (PAP) complex (p <0.01 in both cases). Treatment with dietary n-3FAs concurrently decreased plasma levels of F1.2, D-dimer, and PAP (p <0.05, compared to olive oil controls), implying that the reduction in pain events was related to n-3FA-dependent inhibition of thrombosis. We conclude that dietary n-3FAs reduce the frequency of pain episodes perhaps by reducing prothrombotic activity in sickle cell disease.
High Prevalence of the I278T Mutation of the Human Cystathionine β-Synthase Detected by a Novel Screening Application
Authors: Linnebank Michael , Homberger Anja , Junker Ralf , Nowak-Goettl Ulrike , Harms Erik , Koch Hans
Published: March 2018

Classical homocystinuria due to cystathionine beta-synthase deficiency is one of the disorders revealing a high risk of thromboembolic events and vascular disease. This autosomal-recessively inherited metabolic disorder is considered to be rare with an estimated prevalence of 1:130,000 in the German population. In this study, we developed a novel multiplex PCR generating allele specific fragment lengths to analyse individual genotypes of the two most frequent cystathionine beta-synthase alterations, the I278T mutation, which is worldwide found on up to the half of homocystinuric alleles, and the adjacent polymorphism 844ins68. Screening of 200 unrelated German control subjects revealed a frequency of heterozygosity of 1.5% for I278T corresponding to a calculated frequency of homozygosity of 1:17.800. Our data indicate that homocystinuria due to cystathionine β-synthase deficiency is a frequently unrecognized disorder resulting in a high risk of thromboembolic events.
Platelet Reactivity in Acute Coronary Syndromes: Evidence for Differences in Platelet Behaviour between Unstable Angina and Myocardial Infarction
Authors: Mathur Anthony , Robinson Monique , Cotton James , Martin John , Erusalimsky Jorge
Published: March 2018

Previous work has shown that P-selectin and mean platelet volume, two markers associated with platelet reactivity, are elevated in acute coronary syndromes. This study investigated the possibility that these markers may define unstable angina (UA) and acute myocardial infarction (MI) as two separate conditions based on platelet behaviour. Mean platelet volume (MPV) was higher in UA patients (n = 15) than in those diagnosed with MI (n = 15) (10.7 ± 0.25 fL, vs. 9.8 ± 0.27 fL, P = 0.005). Platelet count was lower in UA than in MI (215 ± 13 × 109/L vs. 271 ± 20 × 109/L, P = 0.03). The percentage of platelets expressing P-selectin was higher in MI than in UA (9.1 ± 1.9% vs. 4.2 ± 0.85%, P = 0.03). This parameter was positively correlated with MPV in UA (r = 0.5, P = 0.04) but negatively correlated in MI (r = -0.6, P = 0.01), with no correlation for ACS as a whole (r = –0.32, P = 0.1). Our results suggest that in MI there is an acute process of generalised platelet activation that is unrelated to changes in MPV, whereas in UA there is an ongoing process of platelet consumption that leads to an increase in platelet size to compensate for a persistent decrease in platelet count. This study suggests that there is a fundamental difference in platelet biology between these two diseases.
Homozygous and Compound-heterozygous Type I Plasminogen Deficiency Is a Common Cause of Ligneous Conjunctivitis
Authors: Zeitler Petra , Seregard Stefan , Ozcelik Ugur , Anadol Deniz , Luchtman-Jones Lori , Meire Francoise , Mingers Anne-Marie , Schambeck Christian , Kreth Hans Wolfgang , Schuster Volker
Published: March 2018

Severe type I plasminogen deficiency has been recently linked to ligneous conjunctivitis, a rare and uncommon form of chronic conjunctivitis. In this study, eight unrelated ligneous conjunctivitis patients living in different parts of the world were examined. All affected subjects from which plasma was available displayed absent or markedly reduced plasminogen antigen and plasminogen functional activity. Molecular genetic studies of seven patients identified a Lys19 → Glu mutation in two boys in a homozygous state, and in two girls in a compound-heterozygous state in which the second plasminogen gene carried a missense (Arg134 → Lys) and a nonsense mutation (Cys133 → Stop), respectively. A fifth patient was shown to be homozygous for a frameshift mutation in plasminogen exon 14 (Gly565ins-G). In two unrelated subjects with ligneous conjunctivitis no mutations in the plasminogen gene were identified. Our results suggest that the Lys19 → Glu mutation is the most prevalent mutation in the plasminogen gene of patients with ligneous conjunctivitis.
Recruitment of Labelled Monocytes by Experimental Venous Thrombi
Authors: McGuinness C. L. , Humphries J. , Waltham M. , Burnand K. G. , Collins M. , Smith A.
Published: March 2018

Objective. Infusing monocytes that have been stimulated to produce fibrinolytic activators and factors that regulate cell proliferation, migration and maturation, might enhance venous thrombus resolution. The aim of this study was to determine the time course of infused monocyte recruitment into venous thrombus in an appropriate model of this disease. Design and Methods. Thrombus was induced in the inferior vena cava of male Wistar rats using reduced flow (80-90% stenosis). The vessel wall was examined at 1hr by scanning electron microscopy. Resolving thrombi with surrounding vena cava were obtained at 1, 7, 14 and 21 days after induction (n = 8). Sections, taken at 0.5 mm intervals (10-15 sections per thrombus), were stained using haematoxylin, Martius Scarlet Blue and antibodies against monocytes, platelets and fibrin. Sections from human venous thrombi (n = 4) were similarly stained. The area occupied by monocytes (in relative pixel units, RPU) was determined using computer aided image analysis. Peripheral rat blood monocytes were extracted, fluorescently labelled and injected intravenously into 7 rats prior to thrombus induction. Vena cava with thrombus was harvested 1 h, 2, 3, 4, 7, 14 and 25 days after induction and their fluorescence measured. The fluorescent content of the caval wall and thrombus was analysed in greater detail at 2 and 25 days after thrombus induction (n = 4 at each time interval). Results. Experimental thrombi were structurally similar to human thrombus and resolved within 14-21 days. Scanning electron microscopy showed minimal endothelial damage at 1 h with signs of early thrombus formation (platelet, red cell leukocyte and fibrin deposition). Neutrophils were the predominant leukocyte in the thrombus at 1 day, with monocytes making up only 0.3% (0.04% sem) of the area of the thrombus. There was a steady increase in thrombus monocyte content and by 21 days the percentage area of thrombus covered by monocytes had increased by over 35 fold to 11.5% (2.3% sem) (p <0.001). Initially, monocytes appeared around the edge of the thrombus and became more evenly distributed through the thrombus as resolution progressed. Labelled monocytes could be found in the circulation up to 1 week after infusion. The fluorescent content (RPU) of the thrombus increased over 25 days (mean RPU At 2 days 0.012, sem 0.005; mean RPU at 25 days 1.062, sem 0.252, p = 0.008). The number of labelled monocytes in the vessel wall peaked at 2 days and decreased thereafter.
Adenovirus-mediated Expression and Packaging of Tissue-type Plasminogen Activator in Megakaryocytic Cells
Authors: Chuang Joseph , Schleef Raymond
Published: March 2018

Platelets release large quantities of plasminogen activator inhibitor 1 (PAI-1) that plays an important role in maintaining the integrity of fibrin-rich thrombi. In addition, tissue-type plasminogen activator (t-PA), a key physiological regulator of fibrinolysis, has been detected in platelet α-granules at low abundance. This information raises the possibility of enhancing t-PA expression in megakaryocytes as a means to enhance the fibrinolytic properties of platelet α-granules and target PAs directly to fibrin clots. This study was initiated to investigate adenovirus (Ad)-mediated expression and packaging of t-PA into α-granules-like structures in the megakaryocytic cell line MEG-01. Ad/t-PA infection of phorbol myristate acetate (PMA)-differentiated MEG-01 cells increased cellular t-PA levels by 120 fold (1580 ± 130 ng/106 cells at 5 MOI) in comparison to non-or Ad/β-gal-infected cells. Fluorescence-activated cell sorter (FACS) analysis indicates that Ad/t-PA-infected cells yielded a homogenous shift in the t-PA staining profile with a 4-fold shift in mean fluorescence in comparison to non- or Ad/β-gal-infected cells. For the isolation of α-granule-like structures, MEG-01 cell homogenates were fractionated by differential centrifugation and two consecutive Percoll density gradients. Fibrin autography of storage granules revealed a prominent lytic zone at Mr 66 kD comigrating with free t-PA. Quantitative analyses indicate that a 16-fold elevation in t-PA antigen within storage granules in comparison to non- or Ad/β-gal-infected cells. To document the ability of t-PA to be stored in a rapidly-releasable form in these cells, we isolated platelet-like particles from the supernatant of differentiated cells and determined that particles from Ad/t-PA-infected cells display a 4-8 fold enhanced secretion of t-PA following treatment with the classical secretagogue calcium ionophore 23187, ADP, or thrombin. Confocal immunofluorescence microscopy analysis indicates that Ad/t-PA mediated productive expression of t-PA in murine megakaryocytes. These data provide support for the concept of increasing the expression of t-PA in megakaryocytes as a means to alter the hemostatic properties of α-granules.

Abbreviations: Ad, Adenovirus; t-PA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor 1; PMA, phorbol myristate acetate; ELISA, enzyme-linked immunosorbent assay; CM, conditioned media; vWF, von Willebrand factor; ECL, enhanced chemiluminescence; PA, plasminogen activator.
Thrombin-induced MCP-1 Expression Involves Activation of the p22phox-containing NADPH Oxidase in Human Vascular Smooth Muscle Cells
Authors: Viedt C. , Nguyen K. , Beer S. , Kreuzer J. , Busse R. , Görlach A. , Brandes R. P.
Published: March 2018

Activation of vascular smooth muscle cells (VMSC) by thrombin induces the expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1). We investigated in cultured human and rat VSMC whether reactive oxygen species (ROS) derived from the vascular NADPH oxidase contribute to this effect. Exposure of cultured VSMC to thrombin rapidly increased ROS formation, phosphorylation of p38 MAP kinase as well as the expression of MCP-1. Specific inhibition of the p22phox subunit of the vascular NADPH oxidase using either p22phox neutralizing antibody or p22phox antisense oligonucleotides attenuated thrombin-induced ROS generation. Furthermore, thrombininduced p38 MAP kinase activation as well as MCP-1 expression were impaired by antioxidants as well as by p22phox antisense oligonucleotides. Inhibition of p38 MAP kinase diminished the thrombin-induced expression of MCP-1. Conclusion: Thrombin, by activating a p22phoxcontaining NADPH oxidase, elicits ROS generation and activation of p38 MAP kinase in VSMC. The subsequent induction of MCP-1 expression highligts the crucial role of the p22phox-containing NADPH oxidase in thrombin-induced signal transduction in VSMC.
Unbalanced Effects of Dermatan Sulfates with Different Sulfation Patterns on Coagulation, Thrombosis and Bleeding
Authors: Vicente C. P. , Zancan P. , Peixoto L. L. , Alves-Sá R. , Araújo F. S. , Mourão P. A. S. , Pavão M. S. G.
Published: March 2018

We compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate dermatan sulfate containing 2-O-sulfated α-L-iduronic acid and 4-O-sulfated N-acetyl-β-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian dermatan sulfate. In contrast, another invertebrate dermatan sulfate, also enriched in 2-O-sulfated α-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-β-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.
Thiosulfinates Inhibit Platelet Aggregation and Microparticle Shedding at a Calpain-dependent Step
Authors: Brohard-Bohn Brigitte , Pain Sabine , Bachelot-Loza Christilla , Auger Jacques , Rendu Francine
Published: March 2018

Thiosulfinates (TSs) are sulfur compounds generated through the processing of different Allium species with antiplatelet property. To further define this platelet inhibitory effect we studied diallyl-TS (Al2TS), dipropyl-TS (Pr2TS), and dimethyl-TS (Me2TS) on platelet responses. The three TSs inhibited dose-dependent platelet aggregation, with IC50 values of 15 ± 2, 19 ± 2, and 9 ± 1 μM for Al2TS, Pr2TS and Me2TS, respectively. TSs had no effect on the expression of a platelet procoagulant surface, measured by flow cytometry as the binding of annexin V-FITC. They inhibited the microparticle shedding and clot retraction. Since the microparticle shedding is a calpain-activation dependent step, we assessed calpain activation by analysis of autoproteolysis in shorter active forms and by talin proteolysis in the presence of TSs. Calpain activation was inhibited by TSs independently of fibrinogen binding. Thus, TSs represent a new category of platelet inhibitors, acting on cal-pain-induced events.
Moderate Wine and Alcohol Consumption: Beneficial Effects on Cardiovascular Disease
Authors: Parks Dale , Booyse Francois
Published: March 2018

Cardiovascular disease, in particular coronary heart disease (CHD) and associated myocardial infarction (MI), is the leading cause of death among adults in the Western world (1). Although CHD is a complex multi-factorial disease, major insights have been gained in our understanding of the etiology underlying the initiation and progression of CHD. The pathogenesis of CHD and the ensuing atherothrombotic complications resulting in MI, involves the complex and often synergistic interplay between multiple dysfunctional cellular and molecular mechanisms that have been altered through interactions with various environmental and/or systemic factors (i. e. CHD risk factors). Typically, these deleterious effects are exerted at the level of the heart muscle, blood vessels and blood components and result in dysfunction in endothelial cells (ECs), smooth muscle cells, cardiac myocytes, blood cells (platelets and monocytes) and plasma components (lipoproteins, fibrinogen, clotting factors, etc.). These combined effects will then contribute further to the initiation and progression of CHD and eventual MI. Consequently, any systemic factors (such as alcohol or wine components) that will reduce, minimize or inhibit these induced dysfunctions will be expected to reduce the overall risk for cardiovascular disease and CHD-related mortality.
Enhanced Fibrinolytic Potential in Mice with Combined Homozygous Deficiency of α2-antiplasmin and PAI-1
Authors: Collen D. , Lijnen H. R. , Dewerchin M.
Published: March 2018

α2-antiplasmin (α2-AP) and plasminogen activator inhibitor-1 (PAI-1) are the main physiological inhibitors of the plasminogen/ plasmin system in mammalian plasma. In the present study, the relative importance of both inhibitors was evaluated with the use of mice with single or combined deficiency of α2-AP and PAI-1 in the same genetic background. Mice with combined deficiency (α2-AP–/–:PAI-1–/–) are viable, develop normally and are fertile. After amputation of the tail, bleeding times are prolonged (>15 min) in α2-AP–/–:PAI-1–/– mice, as compared to double wild-type or single deficient mice (4.6 to 10 min). Spontaneous lysis after 4 h of intravenously injected 125I-fibrin labeled plasma clots is significantly higher in mice with α2-AP deficiency both in the PAI-1+/+ background (89 ± 2% versus 42 ± 3%; p = 0.002) and in the PAI-1–/– background (83 ± 4% versus 53 ± 5%; p = 0.002). PAI-1 deletion in the α2-AP+/+ or α2-AP–/– background, however, has no significant effect (p = 0.13 or 0.18, respectively). Four hours after endotoxin injection, fibrin deposition in the kidneys is not significantly affected by PAI-1 deletion in mice with α2-AP+/+ or α2-AP–/– background (p = 0.07 and 0.19, respectively). In contrast, α2-AP deletion causes significantly reduced fibrin deposition in the PAI-1+/+ background (p = 0.01). Endotoxin injection causes a dramatic increase in PAI-1 antigen levels in kidney extracts of PAI-1+/+ animals, without effect on α2-AP levels.

Taken together, these data indicate that the higher endogenous fibrinolytic capacity observed in mice with combined deficiency is mainly due to the lack of α2-AP and suggest a less important role for PAI-1.