bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety and Efficacy of Continuous Infusion of a Combined Factor VIII – von Willebrand Factor (vWF) Concentrate (Haemate-P™) in Patients with von Willebrand Disease
Authors: Lubetsky Aharon , Schulman Sam , Varon David , Martinowitz Uri , Kenet Gili , Gitel Sanford , Inbal Aida
Published: March 2018

We studied the safety and efficacy of treatment with continuous infusion of a von Willebrand factor (vWF) concentrate Haemate-P in patients with von Willebrand disease (vWD). Three patients with mild and 5 patients with severe forms of vWD, were treated with continuous infusion of Haemate-P by minipump. The indications for treatment were: to prevent bleeding during 9 surgical procedures or 1 vaginal delivery in 6 patients and to treat 2 bleeding episodes in 2 patients. The patients were monitored daily for factor VIII (FVIII:C) and ristocetin cofactor (vWF:RCo) levels and the infusion rate was adjusted to maintain the desired therapeutic level of vWF:RCo. The treatment was effective in preventing surgical bleeding and controlling bleeding episodes. All factor VIII:C and most of the vWF:RCo levels measured during the study period were above the target therapeutic levels. A significant decrease in clearance of FVIII:C and vWF:RCo was observed over the treatment period. Haemate-P consumption averaged 24.3 ± 7.9 vWF:RCo U/kg/day which is approximately half the expected dose had intermittent bolus injections been used. We suggest that continuous Haemate-P infusion is superior to intermittent bolus injections for the treatment of vWD patients by virtue of its efficiency, simplicity and considerable savings.
Fibrinolytic Activity in Peripheral Atherosclerosis in the Elderly
Authors: van der Bom Johanna , Bots Michiel , Haverkate Frits , Meyer Piet , Hofman Albert , Kluft Cornelis , Grobbee Diederick
Published: March 2018

Increased concentrations of plasminogen activator inhibitor type 1 (PAI-1) and of D-dimer have jointly been found in subjects with cardiovascular disease. To understand this apparent paradox of increased inhibition of fibrinolysis (high PAI-1) combined with increased fibrinolytic activity (high D-dimer), we examined the relation between D-dimer, PAI-1 and the activator of fibrinolysis, tissue type plasminogen activator (t-PA) in subjects with varying severity of peripheral atherosclerosis. In 325 subjects selected from the Rotterdam Study, a cohort of 7983 men and women aged 55 years and over, the ankle to brachial systolic blood pressure ratio, t-PA antigen and activity, PAI-1 antigen and D-dimer were measured.

T-PA antigen and t-PA activity were, independent from each other, increased with degree of atherosclerosis; t-PA antigen increased with 3.5 ng/ml (SE 1.7, p = 0.04) and t-PA activity with 0.46 IU/ml (0.20, p = 0.02) per unit decrease in ankle to brachial pressure ratio (i.e. increase in atherosclerosis). PAI-1 antigen was not related to atherosclerosis. More marked atherosclerosis was associated with increased D-dimer, mainly in subgroups with PAI-1 antigen below 50 ng/ml, t-PA antigen below 10 ng/ml, or t-PA activity above 1.5 IU/ml. In contrast to current beliefs, we found that only a fraction of the variation of t-PA antigen was due to the variation in circulating PAI-1 antigen. A slight positive association was observed between t-PA antigen and D-dimer. PAI-1 and t-PA activity were not associated with D-dimer concentration.

In conclusion, in subjects with peripheral atherosclerosis PAI-1 antigen is not increased, but low PAI-1 levels (and possibly also low levels of t-PA antigen and high levels of t-PA activity) appear to be required to increase circulating D-dimer. This suggests that increased D-dimer levels in subjects with atherosclerosis do not reflect increased inhibition, but rather reflect increased fibrinolysis.
A New Animal Model of Thrombophilia Confirms that High Plasma Factor VIII Levels Are Thrombogenic
Authors: Kawasaki Tomihisa , Kaida Takehiro , Arnout Jef , Vermylen Jos , Hoylaerts Marc
Published: March 2018

The thrombotic risk associated with elevated plasma levels of clotting factor VIII (FVIII) was investigated in a mouse model of thrombophilia. After the intravenous injection of recombinant human FVIII and/or of purified FVIII-free human von Willebrand factor (vWF), a controlled mild injury was inflicted on the carotid artery of FVB mice by irradiation with filtered green light in combination with intravenous injection of the dye rose bengal. Formation of a platelet-rich thrombus was continuously monitored for 40 min via transillumination and the thrombus size was measured via image analysis. Administration of recombinant human FVIII at 40 g/kg led to initial FVIII plasma activities equivalent to 250% of normal human plasma FVIII activity and significantly enhanced thrombus size. Immunohistochemical staining illustrated the accumulation of FVIII within the thrombi. Human vWF, even at 10 mg/kg, had no effect on thrombus formation. The thrombotic tendency induced by FVIII was significantly inhibited by the administration of human vWF in a dose-dependent manner. Separate plasma measurements revealed that human FVIII has comparable affinities for human and murine vWF but that human vWF does not effectively bind murine platelets. The inhibition by human vWF of the thrombotic tendency induced by human FVIII could therefore be explained by a lack of accumulation of FVIII within the developing thrombus because of the reduced affinity of human vWF for murine platelets and the reduced occupancy of murine von Willebrand factor by human FVIII after injection of human vWF. These results show that vWF actively participates in FVIII accumulation in the arterial thrombus and provide experimental evidence for epidemiological findings that elevated plasma FVIII levels are associated with an increased thrombotic risk, also in arteries.
Safety of Low-Molecular-Weight Heparin in Pregnancy: A Systematic Review
Authors: Sanson Bernd-Jan , Lensing Anthonie , Prins Martin , Ginsberg Jeffrey , Barkagan Zinoviy , Lavenne-Pardonge Edith , Brenner Benjamin , Dulitzky Mordechay , Nielsen Jørn , Boda Zoltan , Mac Gillavry Melvin , Hamulyák Karly , Theunissen Ingrid , Hunt Beverley , Büller Harry , Turi Susanna
Published: March 2018

Unfractionated heparin (UFH) remains the anticoagulant of choice during pregnancy. Low-molecular-weight heparins (LMWH) are an attractive alternative to UFH due to their logistic advantages and their association with a lower incidence of osteoporosis and HIT. We reviewed all published clinical reports concerning the use of LMWH during pregnancy. In addition, participants of an international interest group contributed a cohort of pregnant women treated with LMWH. Pregnancies were divided into two groups; those with and those without maternal comorbid conditions. The number of adverse fetal outcomes and the occurrence of maternal complications were evaluated in the two groups. In the group of women with comorbid conditions (n = 290), 13.4% of the pregnancies were associated with an adverse fetal outcome. In contrast, in the group of women without comorbid conditions (n = 196), 3.1% were associated with an adverse outcome, which is comparable to that seen in the normal population. We conclude that LMWH appear to be a safe alternative to unfractionated heparin as an anticoagulant during pregnancy.
Factor V Leiden, Prothrombin 20210 G → A and the MTHFR C677T Mutations in Childhood Stroke
Authors: Chalmers E. A. , Thomas A. , Sproul A. , Healey C. , Rafferty I. , McWilliam R. , Eunson P. , McColl M. D.
Published: March 2018

Ischaemic stroke is a rare occurrence in children and in a proportion of cases the aetiology remains unknown. We have investigated the role of thrombophilia in the aetiology of this condition. Of 50 cases identified at two centres, 37 were available for detailed haematological analysis. No cases were identified with deficiencies of antithrombin, protein C or protein S. One case had elevated IgG anticardiolipin antibodies at low titre. The prevalence of the prothrombin 20210 G→A mutation, factor V Leiden (FVL) mutation and the C677T mutation in the MTHFR gene was compared in cases to that observed in random unselected cord blood controls. The odds ratio for stroke was not significantly increased in carriers of the prothrombin mutation (OR 1.2; 95% CI 0.1-10.7), FVL (OR 2.5; 95% CI 0.5-13.5), or the C677T mutation (OR 1.7; 95% CI 0.6-4.5). Our findings suggest that thrombophilia may not play a significant role in the aetiology of stroke in children, although a large prospective study is required to investigate this area further.
Factor V Q506 (Resistance to Activated Protein C) and Prognosis after Acute Coronary Syndrome
Authors: Hillarp Andreas , Zöller Bengt , Erhardt Leif , Berntorp Erik , Dahlbäck Björn , Holm Johan
Published: March 2018

Factor V:Q506 causing resistance to activated protein C (APC-resistance), is a risk factor for venous thrombosis. Some studies have indicated an association with arterial disease, especially in women. We investigated the prevalence of the FV:Q506 allele prospectively in 295 patients with acute coronary syndrome. Mortality and myocardial infarction rate were evaluated after 30 days and after 2 years. The FV:Q506 allele was found in 38 patients. In a Cox proportional hazards model, smokers carrying FV:Q506 had a higher risk of infarction or death within 30 days, compared to non-smokers with a normal genotype (relative risk 2.9 [95% CI 1.2-7.0]). The difference remained significant after 2 years (relative risk 2.8 [95% CI 1.2-6.5]). The effect of the FV:Q506 allele on clinical outcome in acute coronary syndrome has not previously been described. Our results demonstrate a gene-environment interaction between smoking and the FV:Q506 allele, with an increased risk of early complications after an acute ischemic event.
Long-term Management of Homozygous Protein C Deficiency: Replacement Therapy with Subcutaneous Purified Protein C Concentrate
Authors: Sanz-Rodriguez C. , Gil-Fernández J. J. , Zapater P. , Pinilla I. , Granados E. , de Soria V. Gómez-G. , Cano J. , Sala N. , Fernández-Rañada J. M. , Gómez Gómez N.
Published: March 2018

We present the case of a full-term newborn in whom purpura fulminans developed shortly after birth. A diagnosis of homozygous protein C deficiency was established based upon undetectable plasma protein C activity and antigenemia in the newborn infant, and was later confirmed by protein C gene analysis. Specific replacement therapy with intravenous protein C concentrate was started 9 days after birth. This rapidly led to the complete regression of cutaneous lesions and consumption coagulopathy. After stabilization, oral anticoagulation was initiated in association with prophylactic treatment with intravenous protein C concentrate. However, oral anticoagulation was finally abandoned as the patient presented several thrombotic and hemorrhagic episodes clearly related to difficulties with anticoagulation. Due to the hazards related to prolonged venous access, we are currently using subcutaneous infusion of protein C concentrate for the longterm management of this condition, with satisfactory results.
Case-control Study of the Frequency of Thrombophilic Disorders in Couples with Late Foetal Loss and no Thrombotic Antecedent
Authors: Quéré Isabelle , Monpeyrou Françoise , Mercier Eric , Ripart-Neveu Sylvie , Tailland Marie-Laure , Hoffet Méderic , Berlan Jacques , Daurès Jean-Pierre , Marès Pierre , Gris Jean-Christophe
Published: March 2018

Background: Women with familial thrombophilia have an increased risk of still birth. We postulated that the presence of asymptomatic risk factors for venous thrombosis might be a risk factor for late foetal loss. Methods: We performed a case-control study on the prevalence of heritable thrombophilic defects, of antiphospholipid-related markers and of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with at least one episode of late unexplained foetal loss and in control women with successful pregnancies. Partners of cases and controls were also studied. Written conclusions of the pathological examination of the placentas, when available, were also reviewed. Results: We found at least one positive biological risk factor for venous thrombosis in 21.1% of the patients and in 3.9% of the controls (p <10–4). In women, the crude odds ratio for still birth associated with any positive biological risk factor for venous thrombosis was 5.5, 95% confidence interval (95%CI) [3.4-9.0]. No difference was found between partners of cases and controls (5.2% and 4.7%). Using conditional logistic regression analysis, 4 adjusted risk factors for still birth remained: protein S deficiency, positive anti β2 glycoprotein I IgG antibodies, positive anticardiolipin IgG antibodies and the factor V Leiden mutation. The C677T mutation in the MTHFR gene was not an individual risk factor but an homozygous genotype was strongly associated with the former 4 risk factors (16.8% of patients vs. 0.9% of controls). In women with such associations, still births always occurred in absence of folic acid supplementation during pregnancy. Available conclusions of pathological analysis of placentas were found to have a very high proportion of “maternal vascular disease of the placenta” in patients with at least one positive risk marker for thromboembolism, specially in case of association with the C677T MTHFR homozygous genotype, compared to patients with negative markers (p <10–4). Conclusions: Late foetal loss, through placenta thrombosis, may sometimes be the consequence of a maternal multifactorial prothrombotic state associating traditional heritable or acquired thrombosis risk factors to conditions predisposing to an acute mild hyperhomocysteinaemia (coexistence of a genetic predisposition with late pregnancy-related increased folate needs).
Activated Protein C Resistance and the FV:R506Q Mutation in a Random Population Sample
Authors: Rumley Ann , Woodward Mark , Reid Evan , Rumley Joseph , Lowe Gordon
Published: March 2018

Activated protein C (APC) resistance, defined as a low APC ratio, is associated with the factor V mutation R506Q (factor V Leiden). APC ratio may also be influenced by other clinical and coagulation variables, which we studied in 460 men and 495 women aged 25-74 years, from a random population sample (Glasgow MONICA Survey). APC ratio correlated positively with APTT; and inversely with factor VIIIc, factor IXc, antithrombin activity, prothrombin F1+2 fragment, and thrombinantithrombin complexes; but not with other coagulation variables. APC ratio decreased with age, but APTT did not. APC ratio and APTT were significantly lower in women versus men, and were significantly lower in users of oral contraceptives or hormone replacement therapy. The FV:R506Q mutation (prevalence 2.5%) was associated with lower APC ratio and protein C and S activities and with higher factor VIIIc levels; but not with increases in F1+2 fragment or thrombin-antithrombin complexes. APC ratio correlated inversely with total cholesterol and diastolic blood pressure; and in women with triglycerides, systolic blood pressure, and body mass index. Obesity was associated with a significantly lower APC ratio. In contrast, smoking markers correlated positively with APC ratio in men. These associations of APC ratio may be relevant to the increased risks of venous thrombosis with age, female sex, oestrogen use, obesity and high factor VIIIc levels. The association of APC resistance with elevated plasma levels of coagulation markers suggests that this phenotype represents an in vivo hypercoagulable state.

Current address: Dr. E. Reid, Department of Medical Genetics, Addenbrooke’s Hospital, Cambridge, UK
The High Prevalence of Thermolabile 5-10 Methylenetetrahydrofolate Reductase (MTHFR) in Italians Is not Associated to an Increased Risk for Coronary Artery Disease (CAD)
Authors: Sardi Iacopo , Pepe Guglielmina , Marcucci Rossella , Brunelli Tamara , Prisco Domenico , Fatini Cinzia , Capanni Monia , Simonetti Ignazio , Gensini Gian Franco , Abbate Rosanna
Published: March 2018

Mild hyperhomocysteinemia was found to be related to venous thrombosis, cerebrovascular and coronary artery disease (CAD). Some recent studies suggested that a mutation in the gene encoding for 5-10 methylenetetrahydrofolate reductase (MTHFR), due to a transition C→ at nucleotide 677, is a genetic risk factor for vascular disease. However, several further studies could not confirm this association.

We investigated 84 patients with CAD who underwent percutaneous transluminal coronary angioplasty (PTCA) and 106 healthy subjects.

The prevalence of the mutated homozygous genotype was much higher than in other Italian populations, Europeans or other major human groups, but no excess of the Val/Val homozygotes was found in patients (28.5%) with respect to healthy subjects (30.2%). Mutated homozygous MTHFR genotype (+/+) was not found to be related to the clinical manifestations of CAD, to the prevalence of the common risk factors and to the rate of restenosis.

In conclusion, thermolabile MTHFR does not appear to be associated “per se” with the risk for CAD or for restenosis after PTCA. The high frequency of the +/+ genotype in our Italian population (from Tuscany) confirms a wide macroheterogeneity and suggests a microheterogeneity in the genotype frequencies of the different ethnic populations.

Preliminary data of this paper were selected for an Oral Communication at the XVIth ISTH Congress.
A New Direct, Fast and Quantitative Enzyme-linked Ligandsorbent Assay for Measurement of Free Protein S Antigen
Authors: Giri Tusar Kanti , Hillarp Andreas , Härdig Ylva , Zöller Bengt , Dahlbäck Björn
Published: March 2018

A new method to determine the concentration of free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca2+ dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of calcium in the assay helped prevent dissociation of the C4BP-protein S-HPS 54 complex. Three different experiments demonstrated the assay to be specific for free protein S. First, near-identical dose response curves were obtained with protein S in plasma and with purified protein S. Second, addition of purified C4BP to normal plasma resulted in loss of free protein S. Third, protein S depleted plasma gave zero values and around 80% of purified protein S added to protein S depleted plasma, and approximately 70% of protein S added to protein S deficient plasma samples, was recovered with the assay. The assay is fast (involves only a single incubation step of 30 min), sensitive and the range of measurement is 3% to 200% of free protein S when plasma dilution 1 : 20 represents 100%. Intra- and inter-assay coefficients of variation at two levels were 2.3-4.3% and 5.1-7.4%, respectively. In a large protein S deficient family, the assay showed 100% sensitivity and specifity for the causative mutation. Moreover, free protein S levels in anticoagulated protein S deficient patients were completely separated from those obtained in non-anticoagulated controls. The new assay for free protein S is suitable for automation and it provides a useful means for routine clinical purposes to detect protein S deficiencies.
Association of the Platelet Glycoprotein IIIa PIA1/A2 Gene Polymorphism to Coronary Artery Disease but not to Nonfatal Myocardial Infarction in Low Risk Patients
Authors: Humme Jörg , Stricker Jürgen , Nguyen Quoc , Katz Norbert , Philipp Monika , Tillmanns Harald , Hehrlein Friedrich Wilhelm , Rau Matthias , Haberbosch Werner , Gardemann Andreas
Published: March 2018

Background. The platelet membrane glycoprotein IIb/IIIa functions as a receptor for fibrinogen and von Willebrand factor during platelet aggregation. In a small case-control study, evidence has been presented that the PlA2 allele of the platelet glycoprotein GPIIIa PlA1/A2 gene polymorphism might be an independent risk factor for acute myocardial infarction (MI). Methods and Results. We explored the association of the PlA1A2 to the severity of coronary artery disease (CAD), as assessed angiographically in 2252 male individuals, and to myocardial infarction (MI). The severity of coronary heart disease (CHD) was also estimated by calculating a CHD score according to Gensini. The PlA genotype was determined by allele specific restriction digestion. Relation of the PlA2 allele to CAD: In the total population, the frequency of the PlA2 allele was not associated to the presence or to the extent of CAD. Also the CHD scores of PlA1/PlA2 genotypes were essentially the same. However, after exclusion of individuals with high BMI (≥26.9 kg/m2) and/or low apoAI (<1.43 g/l) PlA2PlA2 carriers had clearly higher CHD scores than PlA1PlA1 genotypes; PlA1PlA2 heterozygotes had intermediate values (p <0.05). After division of the study population into one group of individuals without any angiographic signs of CAD (CHD score = 0) and into another group of patients with severe CAD (CHD score (≥120), a strong association of the PlA2 allele with severe CAD was also found in the same low risk groups; e.g. exclusion of persons with high BMI and low apoAI resulted in an Odds ratio of 5.37 (1.46-19.7) (p <0.02). Relation of the PlA2 allele to MI: No association was found between PlA1/PlA2 genotypes and risk of MI neither in the total population nor in low risk subgroups.

Conclusions. Whereas no difference in the distribution of allele and genotype frequencies between controls and survivors of MI could be detected, the PlA2 allele is associated with CHD in low risk patients.
Coronary Thrombosis and the Platelet Glycoprotein IIIA Gene PLA2 Polymorphism
Authors: Durante-Mangoni Emanuele , Ahmed Nabeel , Ruggiero Giuseppe , Tuddenham Edward , Davies Graham
Published: March 2018

Myocardial infarction results from a platelet-rich occlusive coronary thrombus. Platelet membrane glycoprotein IIb/IIIa plays an important role in platelet adhesion and aggregation. Two polymorphisms of the gene encoding the IIIa subunit, PLA1 and PLA2, have been identified. We investigated the frequency of these polymorphisms in 114 consecutive patients with a history of angina-like chest pain admitted for coronary arteriography. Forty-three of these patients had previously suffered a myocardial infarction. The PLA2 polymorphism was found in 21% of the patients with previous myocardial infarction and in 27% of the patients with angina-like chest pain but no previous myocardial infarction (p = 0.634). There was also no significant association with the extent of coronary disease. There is no evidence, therefore, from this study of an association between the PLA polymorphisms and the occurrence of myocardial infarction.
Plasma Lp(a) Levels Are Increased in Patients with Chronic Thromboembolic Pulmonary Hypertension
Authors: Ignatescu M. , Zorn G. , Kneussl M. , Maurer G. , Lang I. M. , Huber K. , Kostner K.
Published: March 2018

Chronic thromboembolic pulmonary hypertension (CTEPH) is a disease resulting from the thromboembolic obstruction of the segmental and/or large size pulmonary arteries, subsequently leading to pulmonary arterial hypertension. Incomplete resolution of acute pulmonary emboli and thrombus organization are believed to be important for the development of the disease. Primary pulmonary hypertension (PPH) is a further disease that at present is poorly understood but shows a clinical picture similar to CTEPH. Since lipoprotein(a) [Lp(a)], a genetically determined risk factor for atherosclerosis and thrombosis, has been found increased in plasma of patients with deep vein thrombosis and pulmonary embolism, we measured plasma Lp(a) levels in 40 patients with CTEPH and 50 patients with PPH and compared them to 50 matched controls. The median for Lp(a) plasma levels was significantly higher in CTEPH patients (26.6 mg/dl) than in PPH patients (9.6 mg/dl) and controls (7.2 mg/dl). Increased plasma Lp(a) could, therefore, play a significant role in the mechanisms of ongoing thrombosis and thrombus organization in CTEPH, while its possible role in PPH can be limited to a small number of patients.
GB Virus C/Hepatitis G Virus Isolates in Japanese Haemophiliacs and their Origins
Authors: Fukuda Yoshihide , Hayakawa Tetsuo , Takamatsu Junki , Saito Hidehiko , Okamoto Hiroaki , Toyoda Hidenori
Published: March 2018

Japanese haemophiliacs have been at high risk for infection with parenterally-transmissible viruses through the use of blood products, especially imported ones. Recently, novel transfusion-transmissible virus, GB virus C (GBV-C)/hepatitis G virus (HGV) were isolated. We investigated the origin and route of transmission of GBV-C/HGV isolates in haemophiliacs in Japan. GBV-C/HGV RNA was measured by nested reverse transcription polymerase chain reaction in 91 Japanese haemophiliacs. Phylogenetic analysis and genotypic grouping of GBV-C/HGV isolates in Japanese haemophiliacs were performed based on sequences in the 5’ untranslated region, and the characteristics were compared with those of reported isolates. GBV-C/HGV infection was present in 19 of 91 haemophiliacs (20.9%). Sequence analysis showed that 15 of the 19 isolates (78.9%) showed sequence similarity to a group in which mainly West African isolates have been reported. The other 4 isolates (21.1%) showed sequence similarity to Asian isolates. None of the GBV-C/HGV isolates showed sequences similar to those generally found in isolates from USA and Europe. The majority of GBV-C/HGV isolates found in Japanese haemophiliacs who are considered to have been infected by imported blood products were similar to those detected in West Africa.
Factor Xa Cleavage of Tissue Factor Pathway Inhibitor Is Associated with Loss of Anticoagulant Activity
Authors: Salemink Irene , Franssen Jo , Willems George , Hemker Coenraad , Li Anguo , Wun Tze-Chein , Lindhout Theo
Published: March 2018

Tissue factor : factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa : TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa : TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa : TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Arg199-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa : TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa : TFPI towards TF : factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF : factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.
A Genetic Propensity to High Factor VII Is not Associated with the Risk of Myocardial Infarction in Men
Authors: Doggen C. J. M. , Cats Manger , Bertina R. M. , Vandenbroucke J. P. , Reitsma P. H. , Rosendaal F. R.
Published: March 2018

Several studies have examined the relation between factor VII and coronary artery disease by measuring factor VII levels in plasma and some found an association between high levels and disease. This suffers problems of interpretation concerning the causality of high factor VII levels, because factor VII levels may be affected by atherogenic risk factors and may become elevated as a consequence of atherosclerosis. We investigated the association between a genetic variant (353Arg→Gln), shown to be related to factor VII levels, and myocardial infarction in a large case-control study, including 560 cases and 644 controls. Individuals carrying the 353Arg-Arg genotype seemed to have a lower risk of myocardial infarction (odds ratio 0.80 [95% confidence interval 0.60-1.06]). In this study, we confirmed higher factor VII antigen and activity level in 529 men homozygous for the 353Arg allele compared with 115 men carriers of the 353Gln allele (around 20% higher).

Our results indicate that a genetic propensity to high factor VII levels is not associated with the risk of myocardial infarction. Since we confirmed the association of the 353Arg-Arg genotype with higher factor VII levels, we conclude that high levels of factor VII are not a causal determinant of myocardial infarction.
Evidence that Adenosine Diphosphate Can Activate Adenylate Cyclase via Conversion to Adenosine in Platelet-rich Plasma Containing Magnesium
Authors: Heptinstall S. , Glenn J. R.
Published: March 2018

When adenosine diphosphate (ADP) is added to hirudinized platelet-rich plasma (PRP) in which the level of platelet cAMP has been pharmacologically elevated, there is an initial rapid fall in the level of cAMP brought about by inhibition of adenylate cyclase. This may be followed by a subsequent activation of adenylate cyclase that does not occur when citrated PRP is used in place of hirudinized PRP, and is more pronounced in the presence of added Mg2+. Here we provide evidence that a) the Mg2+-dependent activation of adenylate cyclase seen in hirudinized PRP is mediated by adenosine, b) the adenosine produced synergizes with forskolin and with DN9693 to raise the level of cAMP in platelets, but not with iloprost, c) Mg2+ does not influence directly the rate or extent of cAMP production and so is more likely to influence the rate of adenosine production, and d) activation of adenylate cyclase by adenosine can lead to inhibition of platelet aggregation. ARL 66096, a P2T purinoceptor antagonist which inhibits ADP-induced platelet aggregation, prevented inhibition of adenylate cyclase by ADP. Conversely, ARL 66096 did not appear to inhibit conversion of ADP to adenosine and subsequent activation of adenylate cyclase.
Heterogeneity and Immunochemical Properties of Anti-β2-glycoprotein I Autoantibodies
Authors: Regnault V. , Hachulla E. , Darnige L. , Roussel B. , Bensa J. C. , Arvieux J.
Published: March 2018

Most anticardiolipin antibodies (ACA) associated with antiphospholipid syndrome (APS) are directed against epitopes expressed on β2-glycoprotein I (β2GPI). Despite a good correlation between standard ACA assays and those using purified human β2GPI as the sole antigen, some sera from APS patients only react in the latter. This is indicative of heterogeneity in anti-β2GPI antibodies. To characterize their reactivity profiles, human and bovine β2GPI were immobilized on γ-irradiated plates (β2GPI-ELISA), plain polystyrene precoated with increasing cardiolipin concentrations (CL/β2GPI-ELISA), and affinity columns. Fluid-phase inhibition experiments were also carried out with both proteins. Of 56 selected sera, restricted recognition of bovine or human β2GPI occurred respectively in 10/29 IgA-positive and 9/22 IgM-positive samples, and most of the latter (8/9) were missed by the standard ACA assay, as expected from a previous study. Based on species specificity and ACA results, IgG-positive samples (53/56) were categorized into three groups: antibodies reactive to bovine β2GPI only (group I) or to bovine and human β2GPI, group II being ACA-negative, and group III being ACA-positive. The most important group, group III (n = 33) was characterized by (i) binding when β2GPI was immobilized on γ-irradiated polystyrene or cardiolipin at sufficient concentration (regardless of β2GPI density, as assessed using 125I-β2GPI); (ii) and low avidity binding to fluid-phase β2GPI (Kd in the range 10–5 M). In contrast, all six group II samples showed (i) ability to bind human and bovine β2GPI immobilized on non-irradiated plates; (ii) concentration-dependent blockade of binding by cardiolipin, suggesting epitope location in the vicinity of the phospholipid binding site on native β2GPI; (iii) and relative avidities approximately 100-fold higher than in group III. Group I patients were heterogeneous with respect to CL/β2GPI-ELISA and ACA results (6/14 scored negative), possibly reflecting antibody differences in terms of avidity and epitope specificity. Affinity fractionation of 23 sera showed the existence, in individual patients, of various combinations of antibody subsets solely reactive to human or bovine β2GPI, together with cross-species reactive subsets present in all samples with dual reactivity namely groups III and II, although the latter antibodies were poorly purified on either column. Therefore, the mode of presentation of β2GPI greatly influences its recognition by anti-β2GPI antibodies with marked inter-individual heterogeneity, in relation to ACA quantitation and, possibly, disease presentation and pathogenesis.
Abnormal Tyrosine Phosphorylation Linked to a Defective Interaction between ADP and Its Receptor on Platelets
Authors: Maclouf Jacques , Rosa Jean-Philippe , Gallet Carole , Vallès Géraldine , Nurden Paquita , Nurden Alan , Lévy-Toledano Sylviane
Published: March 2018

ADP, a primary stimulus of platelets, binds to one or more populations of receptors on the platelet surface. These receptors are linked to discrete activation pathways. Both G proteins and tyrosine kinases have been implicated in the cellular responses to this agonist. We have studied a patient with a congenital abnormality of ADP-induced platelet aggregation in an effort to gain information on the signalling pathways used by ADP. Immunoblotting with a broadly reactive rabbit antibody recognizing the GTP-binding domain of G protein α-subunits, and with rabbit antibodies specific for Giαl-3, and Gα12 all showed normal reactivity when tested against the patient‘s platelets. The phosphorylation of proteins was studied using an anti-phosphotyrosine MoAb (4G10) and platelets stimulated in a platelet aggregometer with ADP, a thromboxane A2 mimetic (IBOP), TRAP-14-mer peptide and α-thrombin. With normal platelets, a time-dependent phosphorylation of several bands in the 60 to 130 kDa mol. wt. range was observed with all agonists. For the patient, minimal aggregation and little or no phosphorylation of proteins of 80-85 kDa (cortactin), 100-105 kDa and 125-130 kDa were seen in response to ADP. The aggregation and phosphorylation responses were slightly modified in the presence of low doses of thrombin but were normal with high doses. Aggregation and tyrosine phosphorylation were virtually absent with IBOP, a finding reproduced when normal platelets were incubated with IBOP and the CP/CPK ADP scavenging system, thereby underlining the role of ADP in the response to IBOP. Our results show that the ADP receptor pathway deficient in the patient is linked to a selective tyrosine phosphorylation response.
The Antiaggregating and Antithrombotic Activity of Clopidogrel Is Potentiated by Aspirin in Several Experimental Models in the Rabbit
Authors: Dol Frédérique , Bernat André , Falotico Robert , Lalé Alain , Savi Pierre , Herbert Jean-Marc
Published: March 2018

It is unknown whether the addition of aspirin might increase both the efficacy and the potency of clopidogrel, a potent and selective ADP blocker. For that purpose, the efficacy of clopidogrel (1–20 mg/kg, p.o.) administered orally to rabbits alone or in combination with aspirin (0.1–10 mg/kg, p.o.) was determined in several experimental models. A potent synergistic effect of the clopidogrel/aspirin association was demonstrated with regard to collagen-induced platelet aggregation measured ex vivo. Similarly, aspirin potentiated the antithrombotic activity of clopidogrel measured with regard to experimental thrombosis induced by a silk thread or on stents placed in an arteriovenous shunt, thrombus formation following electrical stimulation of the rabbit carotid artery and with regard to 111In-labeled platelet deposition on a stent implanted in an arteriovenous shunt or on the subendothelium following air drying injury of the rabbit carotid artery. A similar potentiating effect of aspirin was obtained with regard to myointimal proliferation (restenosis) in the femoral arteries of atherosclerotic rabbits which occurred as a consequence of stent placement. The clopidogrel/aspirin combination showed only additive-type effects on bleeding time prolongation induced by ear transection in the rabbit, therefore showing that combined inhibition of cyclooxygenase and ADP‘s effects provide a marked enhanced antithrombotic efficacy. Such a combination may provide substantial protection against platelet aggregation leading to thrombotic occlusion at sites of endothelial injuries and coronary artery stenosis in humans.
Control of Smooth Muscle Cell Proliferation – The Role of the Basement Membrane
Authors: Roy Joy , Tran Phan Kiet , Lundmark Karin , Rahman Adnan , Hedin Ulf
Published: March 2018

In atherogenesis and in response to vessel injury, arterial smooth muscle cells (SMCs) are activated from a quiescent, differentiated state into an actively proliferating and synthetic phenotype which migrate into the intima where the cells participate in the formation of a fibrous plaque or intimal hyperplasia. The mechanisms involved in the control of SMC function are not clear and no preventive therapy against SMC activation is available. Interactions between SMCs and the extracellular matrix have been shown to influence SMC structure and function through integrin-mediated signaling processes. The SMC basement membrane is a specific form of extracellular matrix which seems to be crucial for the maintenance of SMC quiesence and the disruption of these interactions is part of cellular activation after atherogenic or traumatic stimuli. This concept of “negative growth control” may constitute a future target for the development of new strategies in the prevention of SMC activation in atherogenesis and restenosis formation.
Gene Regulation and Arteriosclerosis: Are Developmental Programs Reactivated in Vascular Disease?
Authors: Patterson Cam
Published: March 2018

The molecular mechanisms regulating the development of vascular diseases such as atherosclerosis remain poorly understood at present. Similarities between genetic programs observed during the course of vascular disease with those observed during vascular development suggest that developmental processes are recapitulated in vascular disease. The earliest event in vascular development is the differentiation of endothelial cells from their mesodermally-derived hamangioblastic precursors. The receptor for vascular endothelial growth factor, KDR/flk-1, plays a critical role in these earliest stages of vascular development. During development and in the adult, expression of this receptor is restricted to vascular endothelial cells and their immediate precursors. We have therefore endeavored to determine the transcriptional events regulating KDR/flk-1 expression, with the hope of gaining insight into processes of vascular development that might also be important in vascular diseases of the adult.
Soluble Vascular Cell Adhesion Molecule-1 (VCAM-1) as Potential Marker of Atherosclerosis
Authors: Weirich Uta , Nordt Thomas K. , Ruef Johannes , Bode Christoph , Peter Karlheinz
Published: March 2018

An increasing number of descriptive reports on soluble adhesion molecules and association with various diseases are published. Throughout these reports soluble adhesion molecules are identified as markers of inflammation. Since atherosclerosis demonstrates features of a chronic inflammatory disease, a potential association of soluble adhesion molecules with atherosclerosis has been postulated. However, conflicting results have been reported. One reason for this might be the differing definitions of atherosclerosis and patient groups. Besides the definition of atherosclerosis based on clinical symptoms, few reports use a direct quantification of atherosclerosis in their search for a marker of atherosclerosis. In those reports that quantify atherosclerosis, sVCAM-1 seems to be more specific for atherosclerosis than other markers. The serum level of sVCAM-1 appears to correlate with the extent of atherosclerosis and might allow for the detection of early stages of atherosclerosis. Large scale prospective studies will have to prove that sVCAM-1 can be used as a diagnostic tool for the detection of early stages of asymptomatic atherosclerosis and whether an early therapeutic intervention based on this approach is able to prevent progression and manifestation of the clinical sequelae of atherosclerosis.
Lipid Lowering Therapy and Stabilization of Atherosclerotic Plaques
Authors: Müller-Wieland Dirk , Krone Wilhelm
Published: March 2018

Lipid-lowering therapy leads to a great reduction of cardiovascular complications, but has almost no effect on the degree of stenosis of coronary arteries. These and other studies have lead to a new paradigm of coronary artery disease, i. e. clinical prognosis is not only determined by the extent of a single stenosis, but mainly by the number and structure of atherosclerotic plaques. Rupture of an instable or vulnerable plaque, characterized by a large lipid-rich central core, inflammatory cells, and a thin fibrous cap, causes sudden thrombus formation and thereby acute coronary syndromes. There is accumulating evidence that cholesterol lowering can result in plaque stabilization and improvement of endothelial dysfunction.
Fibrinolytic Response to Venous Occlusion Compared to Physical Stress Test in Young Patients with Coronary Artery Disease
Authors: Graf Senta , Beckmann Renate , Hornykewycz Stephan , Koller-Strametz Jeanette , Binder Bernd R. , Nikfardjam Mariam , Huber Kurt
Published: March 2018

Introduction: Venous occlusion (VO) and exercise stress (ES) are stimulators of the fibrinolytic system. Aim of this study was to answer which of both stimulation tests is more useful in patients with symptom-limited coronary artery disease (CAD) to evaluate possible defects in the fibrinolytic system.

Methods and results: We investigated 20 patients (M/F = 15/5; mean age = 36.7 years) with angiographically proven CAD for their plasma levels of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-type-1 (PAI-1) at basal conditions as well as after VO and at maximal ES (standardised bicycle stress test) and compared the data to those obtained from 12 sex- and age-matched healthy controls (M/F = 9/3; mean age = 40.4 years). At basal conditions mean t-PA activity and t-PA antigen plasma levels were within the normal range and comparable between the two study groups. After both VO and maximal ES, mean t-PA activity and t-PA antigen levels increased significantly more in the control group as compared to the CAD group. Mean PAI-1 activity plasma levels were significantly higher in the CAD group at basal conditions before VO (patients 7.0 ± 3.1; controls 3.9 ± 3.9; IU/ml; p = 0.025) as well as before ES (patients 8.1 ± 3.5; controls 4.3 ± 3.8; IU/ml; p = 0.009). PAI-1 activity plasma levels showed a significant decrease for patients and controls only after VO, while PAI-1 activity was not significantly decreased in both study groups at maximal ES.

Discussion: The significantly higher increase in mean plasma levels of t-PA activity and t-PA antigen after VO compared to ES in both groups might be explained by the fact that CAD induced symptoms in the patients during ES thus permitting only 80% of their age, sex, and body mass index related optimal work load.

Conclusion: VO and ES are applicable triggers of the endogenous fibrinolytic system in healthy subjects and patients who are not limited in their physical exercise. Standardised VO appears to be superior to ES as stimulation test of the endogenous fibrinolytic system in patients with symptomatic CAD.
Secondary Prevention of Stroke
Authors: MacWalter R. S.
Published: March 2018

Stroke is a common disorder and a leading cause of disability and death. Ischaemia is a more common cause than haemorrhage and radiological imaging is required to accurately differentiate these. Some specific risk factors for stroke are non-modifiable – these include age, gender, racial and hereditary factors. Certain risk factors for ischaemic stroke can be identified and modification of these can be used for secondary prevention – examples include hypertension, heart diseases, atrial fibrillation, diabetes mellitus, dyslipidaemia, smoking, excessive alcohol consumption and carotid stenosis. Carotid endarterectomy is valuable in selected patients. In ischaemic stroke and transient ischaemic attack antithrombotic therapy is an option used in secondary prevention. In atrial fibrillation, warfarin should be used where possible in secondary prevention. When warfarin is contraindicated aspirin should be used. In other patients, an antiplatelet regime is appropriate – aspirin is commonly used and is the least expensive regime. Other antiplatelet agents such as dipyridamole, ticlopidine and clopidogrel may have a place. Younger patients with ischaemic stroke may have a thrombophilia state and should be appropriately investigated.
Submassive and Massive Pulmonary Embolism: A Target for Thrombolytic Therapy?
Authors: Geibel Annette , Kasper Wolfgang , Konstantinides Stavros
Published: March 2018

Thrombolytic agents have been consistently demonstrated to dissolve pulmonary thrombi much more rapidly and effectively than heparin alone. Rapid resolution of pulmonary embolism (PE) is accompanied by a significant decrease in pulmonary artery pressure and an improvement in right ventricular function. However, it is no longer than 7 days until the findings of patients treated with heparin improve to a similar extent. Previous studies were not designed to determine whether this short-lasting difference in favor of thrombolysis can indeed affect the prognosis of patients with PE and thus justify the 1% (or even higher) risk of cerebral or fatal bleeding. Recently, two large registries demonstrated the importance of right ventricular dysfunction assessed by echocardiography as an independent predictor of mortality. Thrombolytic treatment was shown in one of these registries to be associated with a 50% reduction of death risk in clinically stable patients with right ventricular enlargement. It was thus possible to identify a group of patients with massive PE who are most likely to benefit from early thrombolysis. These findings now have to be confirmed by a prospective randomized trial which will compare thrombolysis with heparin alone in this high-risk patient population, focusing on clinical end points such as overall and event-free survival in the acute phase of PE.
Primary Stenting for Acute Myocardial Infarction
Authors: Yhip A. , Peter J. , Smalling Richard W.
Published: March 2018

Primary stenting for acute MI has been shown to be an improvement over PTCA alone. As with primary PTCA however there is an obligate delay in restoration of TIMI flow due to the time necessary for mobilization of the cath lab team. It is possible that a hybrid approach using partial thrombolysis plus early IIB/IIIA inhibitor administration followed by urgent angiography and stenting of the culprit lesion will be the ideal approach.