bluuebunny/crossref_metadata_2025 huggingface dataset:
Solid Tumors and Factor VIII Antibodies
Authors: Hauser Irene , Lechner Klaus
Published: March 2018

Purpose: To analyze the association between solid tumors and the occurrence of factor VIII antibodies and the response to treatment.

Patients and Methods: Published case reports describing the association of a solid tumor and a factor VIII antibody retrieved from 20 data bases. 40 cases were reported and 27 cases were analyzable.

Results: Factor VIII antibodies occurred in a close temporal relationship with the detection of the tumor in most cases. No association with a specific type of tumor could be identified. Immune suppressive treatment with prednisone ± cyclophosphamide was successful in the majority of cases.

Conclusion: It is likely that there is a causal association between some solid tumors and factor VIII antibodies, but it is an extremely rare complication of cancer. The immunoglobuline nature of the inhibitor and the good response to immune suppressive treatment suggests that it is an autoimmune phenomenon. The pathogenesis is unknown.
Factor XIIa Is a Kinetically Favorable Plasminogen Activator
Authors: Feddersen Karina , Røjkjær Rasmus , Schousboe Inger
Published: March 2018

Initiation of the plasma contact system has been shown to play a significant role in the fibrinolysis, activating both pro-urokinase and plasminogen. The aim of the present study was to further evaluate the functional role of the factor XIIa catalyzed activation of plasminogen. Activation of plasminogen by factor XIIa followed the Michaelis-Menten rate equation. In a continuous assay system the Km was 0.27 μΜ; the kcat 0.078 min−1 and the kcat/Km 0.31 × 106 Μ−1 × min−1. In an endpoint assay system the Km was 0.58 μΜ; the kcat 0.096 min−1 and the kcat/Km 0.16 × 106 Μ−1 × min−1. The discrepancy between the kcat in the two assays is not significant. Theoretically the higher Km in the end-point assay system may be due to the presence or generation of an unidentified competitive inhibitor in this assay system. Comparing the catalytic constants of factor XIIa with those of urokinase activation of plasminogen, the specificity constant, kcat/Km, of factor XIIa activation of plasminogen was 20-fold lower. However, taking the low physiological concentration of urokinase into account, the efficiency of activated factor XII is equivalent to that of urokinase. When monitoring factor XIIa activation of plasminogen in a clot lysis assay, the clot lysis time was 2- to 4-fold as long as that accommodated by urokinase at factor XIIa concentrations equivalent with 5-20% of the zymogen concentration in plasma. The factor XIIa mediated clot lysis was prevented completely by the presence of a polyclonal antibody to factor XII.
Longevity Is Independent of Common Variations in Genes Associated with Cardiovascular Risk
Authors: Andersen-Ranberg Karen , de Maat Moniek , Kristensen Søren Risom , Jeune Bernard , Gram Jørgen , Jespersen Jørgen , Bladbjerg Else
Published: March 2018

Do extremely old persons have a genetically favourable profile which has protected them from cardiovascular death? We have tried to answer this question by measuring DNA polymorphisms of selected cardiovascular risk indicators [factor VII, FVII (R/Q353, intron 7 (37bp)n, and -323ins10), β fibrinogen (-455G/A), plasminogen activator inhibitor type 1, PAI-1 (-675(4G/5G)), tissue plasminogen activator, t-PA (intron 8 ins311), platelet receptor glycoprotein IIb/IIIa, GPIIb/IIIa (L/P33), prothrombin (20210G/A), methylene tetrahydrofolate reductase, MTHFR (A/V114), angiotensin converting enzyme, ACE (intron 16 ins287), and angiotensinogen (M/T235)]. Blood was collected from 187 unselected Danish centenarians, and 201 healthy Danish blood donors, aged 20-64 years (mean age 42 years). Genomic DNA was amplified using PCR and the genotype was determined by RFLP methods or allele-specific amplification followed by agarose gel electrophoresis. The frequencies of the high-risk alleles in centenarians were: for FVII R/Q353 0.91; for FVII intron 7 (37bp)n 0.67; for FVII-323 ins10 0.90; for fibrinogen 0.16; for PAI-1 0.52; for t-PA 0.59; for GPllb/llla 0.16; for prothrombin 0.008; for MTHFR 0.33; for ACE 0.52; and for angiotensinogen 0.36. Comparable frequencies were observed in the blood donors. Subgroup analysis of men and women separately gave similar results. The genotype frequencies in the centenarians and the blood donors were similar for all polymorphisms, and this study suggests that common variations in genes associated with cardiovascular risk do not contribute significantly to longevity.
Collagen or Collagen-related Peptide Cause [Ca2+]i Elevation and Increased Tyrosine Phosphorylation in Human Megakaryocytes
Authors: Melford S. K. , Bunce C. M. , Gibbins J. , Watson S. P. , Mountford J. C.
Published: March 2018

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in mega-karyocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.
The Factor V Gene A4070G Mutation and the Risk of Venous Thrombosis
Authors: Nicaud Viviane , Gandrille Sophie , van Dreden Patrick , Amiral Jean , Aubry Marie-Laurence , Fiessinger Jean-Noël , Emmerich Joseph , Aiach Martine , Alhenc-Gelas Martine
Published: March 2018

The A4070G polymorphism in exon 13 of the factor V (FV) gene, which replaces His by Arg at position 1299 of the B domain, was recently shown to influence circulating FV levels and to contribute to the activated protein C (APC) resistance phenotype. We examined the impact of this polymorphism in a population of unselected patients with venous thromboembolic disease (VTE). The prevalence of the G4070 (R2) allele was determined in 205 patients and 394 healthy subjects of similar age and sex distribution. Thirty-seven patients (18%) were heterozygous for the R2 allele and 1 (0.5%) was homozygous. Forty-four controls (11.2%) were heterozygous for the R2 allele and 1 (0.2%) was homozygous. Thus, the allelic frequency was significantly higher in the patients with VTE than in the healthy controls, with respective values of 9.5% and 5.8%. The odds ratio was 1.8 (95% CI: 1.1-2.8, p = 0.02), pointing to an increased risk of VTE in carriers of the R2 allele. After excluding subjects with putative or confirmed gene defects (mainly the FV R506Q mutation), the R2 allele was still a risk factor for VTE in the remaining patients, with an odds ratio of 2.0 (95% CI: 1.2-3.5, p = 0.01), demonstrating that this polymorphism is itself a risk factor. This study also confirms that the R2 allele influences APC resistance (APCR) in the absence of the FV R506Q mutation.
Low Levels of Heparin-releasable Tissue Factor Pathway Inhibitor in Young Patients with Thrombosis
Authors: Alberio G. , Moia M. , Mannucci P. M. , Ariëns R. A. S.
Published: March 2018

An association between deficiency of tissue factor pathway inhibitor (TFPI) and thrombosis has not been clearly demonstrated in humans, but previous studies have focused on the measurement of plasma TFPI, which is only a small part of the total body TFPI. The major fraction of this natural anticoagulant can be measured in plasma after release by heparin injection. To investigate if deficiency of heparin-releasable TFPI is associated with thrombosis, we measured TFPI activity in plasma before and 10 min after intravenous injection of 7500 IU unfractionated heparin in 64 young patients with venous thrombosis, 49 young patients with arterial thrombosis and 38 healthy individuals. Post-heparin TFPI activity levels were significantly lower in the group of patients with venous thrombosis than in controls (mean ± SD: 230% ± 39 vs 260% ± 34, p = 0.0002), whereas there was no difference for patients with arterial thrombosis. Defining the normal range as the mean ± 2 SD of TFPI activity in controls, twelve patients had low post-heparin TFPI activity levels, seven with venous and five with arterial thrombosis. Low levels of TFPI activity were confirmed by immunoassay in six of the seven patients with venous thrombosis and two of the five patients with arterial thrombosis, and were present also in at least one first degree relative of six patients, suggesting that the defect might be inheritable. However, the causative role of low heparin-releasable TFPI remains uncertain, because co-segregation of the defect with thrombotic symptoms could not be demonstrated in the small number of families studied.
Post-DDAVP Thrombocytopenia in Type 2B von Willebrand Disease Is not Associated with Platelet Consumption: Failure to Demonstrate Glycocalicin Increase or Platelet Activation
Authors: Steffan A. , Pontara E. , Zucchetto A. , Rossi C. , De Marco L. , Girolami A. , Casonato A.
Published: March 2018

Thrombocytopenia is frequently reported in type 2B von Willebrand disease (vWD), and thought to be related to the abnormally high affinity of 2B von Willebrand factor (vWF) for platelet GPIb-IX. To gain an insight into the nature of this thrombocytopenia, we measured plasma glycocalicin (GC) levels (as a marker of platelet turnover), and platelet surface expression of the alpha granule protein P-selectin (as a marker of platelet activation) in 9 patients with type 2B vWD before, and in 4 patients also following the infusion of 1-desamino-8-d-arginine vasopressin (DDAVP). Three patients presented a persistent decrease of platelet counts in the resting condition. GC levels were within the normal range, regardless of the platelet counts, in all but one patient who presented, on the other hand, a normal platelet count. Moreover, platelets expressed normal amounts of P-selectin on their surface, regardless of platelet counts. These findings suggest that the thrombocytopenia observed in type 2B vWD is not due to platelet activation and subsequent consumption in circulation.

Despite a significant, albeit transient, decrease in platelet count, DDAVP did not induce an increase in plasma GC levels, nor enhance P-selectin expression. These observations indicate that the acute post-DDAVP thrombocytopenia in type 2B vWD is not related to platelet activation and consumption. We advance that the post-DDAVP 2B vWF is hemostatically more active, and able to induce agglutination but not aggregation of circulating platelets. This would explain both the prompt recovery of basal platelet counts after the post-DDAVP decrease, and the lack of reported thrombotic complications in this disorder.

Therefore, even though 2B vWF is characterized by an enhanced affinity for the platelet surface, its binding to platelet GPIb-IX in the soluble phase is not able to induce true platelet aggregation; vWF thus appears to be mainly an adhesive protein, rather than an aggregating agent.
Comparison of the Effects of Apo(a) Kringle IV-10 and Plasminogen Kringles on the Interactions of Lipoprotein(a) with Regulatory Molecules
Authors: Green Michael , LoGrasso Philip , Boettcher Brian , Madison Edwin , Curtiss Linda , Miles Lindsey , Xue Song
Published: March 2018

Lipoprotein(a) [Lp(a)] is associated with atherosclerosis and with disease processes involving thrombosis. Lp(a) contains apoprotein (a) [apo(a)], which has a sequence highly homologous to plasminogen. Hence, Lp(a) binds directly to extracellular matrix, cellular plasminogen receptors and fibrin(ogen) and competes for the binding of plasminogen to these regulatory surfaces. These interactions may contribute to the proatherothrombogenic consequences of high Lp(a) levels. These interactions are mediated by lysine binding sites (LBS). Therefore, we examined the role of apo(a) kringle IV-10 [the only apo(a) kringle demonstrated to exhibit lysine binding activity in the intact lipoprotein] in the interaction of Lp(a) with these regulatory molecules. We have compared directly apo(a) KIV-10 with plasminogen K4 to examine whether these highly structurally homologous kringle modules are also functionally homologous. Futhermore, because the plasminogen K5-protease domain (K5-PD) binds directly to fibrin, we have also examined the ability of this plasminogen fragment to inhibit the interaction of Lp(a) with these regulatory molecules and with extracellular matrix. Apo(a) KIV-10 competed effectively for the binding of 125I-Lp(a) to these surfaces but was less effective than either intact Lp(a), plasminogen K4 or plasminogen. Plasminogen K5-PD was a better competitor than apo(a) KIV-10 for 125I-Lp(a) binding to the representative extra-cellular matrix, Matrigel, and to plasmin-treated fibrinogen. In contrast, plasminogen K5-PD did not compete for the interaction of Lp(a) with cells, although it effectively competed for plasminogen binding. These results suggest that Lp(a) recognizes sites in all of the regulatory molecules that are also recognized by apo(a) KIV-10 and that Lp(a) recognizes sites in extracellular matrix and in plasmin-modified fibrinogen that also are recognized by plasminogen K5-PD. Thus, the interaction of Lp(a) with cells is clearly distinct from that with extracellular matrix and with plasmin-treated fibrinogen and the recognition sites within Lp(a) and plasminogen for these regulatory molecules are not identical.

Portions of this manuscript were presented at the 69th Meeting of the American Heart Association, New Orleans, LA 1996.
Induction of Hypercoagulability Condition by Chronic Localized Cold Stress in Rabbits
Authors: Khatun Selina , Belayet Hossain , Tokunaga Naoki , Sumimoto Kazuhiro , Kobayashi Takao , Terao Toshihiko , Kanayama Naohiro
Published: March 2018

To evaluate the effect of cold-induced stress on renal and hepatic blood flow and coagulation parameters, rabbits’ soles were exposed to ice pad (0° C). Renal and hepatic blood flow was measured after 1 h and 15 days of cold stress. Coagulation parameters (0, 8th and 15th days of stress) and histological studies were performed. Renal and hepatic blood flow was significantly reduced after cold-stress. Decreased platelet count, antithrombin III (AT III) activity, increased fibrinogen (Fbg) level, shortened activated partial thromboplastin time (aPTT) and prothrombin time (PT) was found after 8 and 15 days of cold-stress. Histology showed enlarged glomeruli with fibrin deposition in kidney, ischemic changes and fibrin deposition in liver and hemorrhagic necrosis in adrenal cortex. We conclude that undesirable localized cold induced sympathetic stimulation in daily life may be a predisposing factor for coagulopathy.
Enhancement of Protein S Anticoagulant Function by β2-glycoprotein I, a Major Target Antigen of Antiphospholipid Antibodies:
Authors: Wei Zhang Hong , Shen Christine , Butman Bryan , Jeffries Eddie , Lahita Robert , Myones Barry , Merrill Joan
Published: March 2018

Thrombosis in the antiphospholipid syndrome has been associated with acquired deficiency of the anticoagulant protein S. We sought evidence that β2-glycoprotein I, a major target antigen for antiphospholipid antibodies, is involved in regulation of protein S activity. Incubation of purified protein S or plasma with β2-glycoprotein I reversed functional modulation of protein S by its plasma inhibitor, the C4b-binding protein. In a plasma-free ELISA, β2-glycoprotein I prevented the binding of protein S and C4b-binding protein when pre-incubated with immobilized protein S but not when similarly preincu-bated with C4b-binding protein. β2-glycoprotein I in fluid phase interfered with precipitation of protein S by sepharose-bound C4b-binding protein. Effects of β2-glycoprotein I on protein S function were inhibited by one of four monoclonal anti-β2-glycoprotein I antibodies. These data suggest that β2-glycoprotein I helps maintain adequate plasma levels of circulating free, active protein S. Antiphospholipid (anti-β2-glycoprotein I) antibodies might cause sporadic thrombosis, at least in part, by impairing this novel regulatory mechanism.
The Sensitivity and Specificity of Commercial Reagents for the Detection of Lupus Anticoagulant Show Marked Differences in Performance between Photo-optical and Mechanical Coagulometers
Authors: Mackie I. J. , Purdy G. , Machin S. J. , Lawrie A. S.
Published: March 2018

This study was undertaken to appraise the application of those reagents most widely used in the UK for the detection and confirmation of lupus anticoagulant (LA) on an Amelung KC4A and a Sysmex CA-6000™ coagulometer. Five sets of dilute Russell’s viper venom time (DRVVT) reagents were assessed as well as the Textarin®-PL/ Ecarin ratio. Each DRVVT method comprised both LA detection and confirmation reagents provided by the same manufacturer. Samples were obtained from 20 normal healthy subjects, 10 LA-positive patients, 10 patients receiving oral anticoagulant therapy (OAT) who had previously been documented as LA-positive, a further 10 LA-negative patients receiving OAT and 10 LA-negative patients receiving unfractionated heparin therapy.
The sensitivity and specificity of the reagents exhibited considerable variation not only between reagents, but also when the same reagent was used on the two analysers. Sensitivity ranged from 62 to 97% (all reagents both analysers), specificity went as low as 23% (Gradipore reagent on the CA-6000™) and as high as 100% (American Diagnostica Inc on both KC4A and CA-6000™). On the KC4A instrument, Unicorn Diagnostics’ lupus anticoagulant kit offered the best compromise of sensitivity and specificity (sensitivity 83% and specificity 81%). On the CA-6000™ the reagents supplied by American Diagnostica Inc exhibited optimal performance (sensitivity 90% and specificity 100%). The results indicate a need to optimise test reagents for specific analyser types, a procedure which can only be undertaken with preparations such as the proposed NIBSC reference plasmas for the detection of lupus anticoagulant.
Transcriptional Regulation of the Murine Urokinase-type Plasminogen Activator Gene in Skeletal Myoblasts
Authors: Miralles Francesc , Ibáñez-Tallon Inés , Parra Maribel , Crippa Massimo , Blasi Francesco , Besser Daniel , Nagamine Yoshikuni , Muñoz-Cánoves Pura
Published: March 2018

We have previously shown that urokinase-type plasminogen activator (uPA) is highly expressed in murine C2C12 myoblasts and that antibodies against uPA are able to block both myoblast fusion and differentiation. Here we show the characterization of cis-acting elements in the mouse uPA promoter in vitro which are involved in uPA gene expression in C2C12 myoblast cells. DNase I hypersensitive (HS) site analysis revealed the presence of three HS sites in myoblasts. Deletion analysis of stably transfected uPA-promoter constructs revealed that at least two of the three HS sites accounted for the high transcriptional expression in C2C12 cells. One was located at -2.4 kb and corresponded to a known PEA3/AP1A element and the other one was located at -4.9 kb and contained a CArG box and a CRE element. So far, no regulatory function had been assigned to this CRE/CArG element. Both HS sites alone were able to activate transcription of a heterologous promoter and showed a cooperative effect when placed together. Electrophoretic mobility-shift assays using myoblast nuclear extracts and specific antibodies demonstrated that cJun, JunD and ATF2 bound to the PEA3/AP1A element, whereas the CRE/CArG element bound SRF. Altogether, these results suggest that high uPA expression in myoblasts is dependent on the cooperation of two regulatory sites in the uPA promoter.
High Levels of Circulating Thrombomodulin in Human Foetuses and Children
Authors: Aurousseau Marie-Hélène , Gozin Danielle , Daffos Fernand , D’Angelo Armando , Forestier François , Boffa Marie-Claire , Menashi Suzanne
Published: March 2018

Thrombomodulin (TM) is an endothelial cell surface proteoglycan with anticoagulant functions, also implicated in cell proliferation, cell-cell adhesion and differentiation. In this study we determined circulating plasma TM (pTM) levels in human foetuses at different stages of pregnancy, at birth and in childhood. TM levels increased with gestational age, the median level reaching a peak of approximately 165 ng/ml between the 23rd and 26th week, thereafter decreasing gradually, reaching a value of 108 ng/ml at birth. pTM continues to decrease progressively during childhood, reaching in the 5-15 years group a median of 56 ng/ml which approaches the adult value. The pTM peak was statistically significant and represents a specific foetal phenomenon as it was independent of the corresponding maternal values. As a whole, the pTM pattern during foetal maturation appears totally different from that of protein C, prothrombin and other coagulation activators and inhibitors and thus, TM may play in the foetus another role in addition to its well-known anticoagulant function.
Correction for Lack of Coincidence of Normal and Abnormal Calibration Slopes in ISI Determination
Authors: Besselaar A. M. H. P. , Jespersen J. , Tripodi A. , Houghton D. , Poller L.
Published: March 2018

An underlying assumption of the WHO orthogonal regression model for prothrombin time standardisation is that a single line describes the relationship between PT of abnormal and normal plasmas. The aim was to evaluate Tomenson’s correction for lack of coincidence using the model of the ECAA human reagent with lyophilized plasma calibrations. Local ISI calibrations using ECAA lyophilized normal and abnormal plasmas were performed on coagulometers with the ECAA human thromboplastin. They gave a high incidence of non-linearity of the two calibration slopes. The mean percentage deviation from assigned values of the target coumarin plasmas coagulometer INR at 45 centres before local ISI correction was 16.6% and 11.9% respectively in two studies. After local ISI calibration, the unsigned deviation was reduced to 5.54% and 6.61% respectively. Tomenson’s correction for non-linearity further reduced the mean deviation to 3.08% and 2.96 % respectively in the two studies demonstrating the value of the procedure.
Endogenous Nitric Oxide Acts as a Natural Antithrombotic Agent In Vivo by Inhibiting Platelet Aggregation in the Pulmonary Vasculature
Authors: Emerson Michael , Momi Stefania , Paul William , Francesco Paolo , Page Clive , Gresele Paolo
Published: March 2018

Nitric oxide (NO) is a powerful vasodilator and an inhibitor of platelet aggregation in vitro. While the ability of NO to modulate vascular tone in vivo has been proven, only a few studies have assessed its platelet inhibitory activity in vivo.

We have employed two complementary animal models of pulmonary platelet thromboembolism to assess the antithrombotic activity of endogenous NO in vivo.
The inhibition of nitric oxide synthase (NOS) by L-NAME significantly potentiated while the administration of the NOS substrate L-arginine significantly reduced the accumulation of 111In-labelled platelets in the pulmonary vasculature of rabbits induced by intravenous collagen plus epinephrine. L-NAME or L-arginine did not, however, modify 111In-labelled erythrocyte distribution in lungs and phenylephrine had no effect on platelet accumulation following collagen + adrenaline, suggesting that the effects of L-NAME were not due to vasoconstriction but rather to a direct modification of platelet function. In mice, L-NAME significantly reduced the dose of collagen + adrenaline required to induce thromboembolic mortality, increased the fall in circulating platelets and increased the % of pulmonary vessels occluded by platelet thrombi. The effects of L-NAME were reversed by L-arginine but not by a dose of nicardipine exerting maximal vasodilatation. Phenylephrine did not potentiate collagen + adrenaline-induced mortality.

In the pulmonary vasculature in vivo, endogenous NO inhibits collagen + adrenaline-induced aggregation and enhances platelet disaggregation. This natural modulator function of NO is exerted via a direct effect on platelets and not as a result of haemodynamic changes.
Thrombin Receptor Occupancy Modulates Aggregation Efficiency and Platelet Surface Expression of vWF and Thrombospondin at Low Thrombin Concentrations
Authors: Legrand Chantal , Kasirer-Friede Ana , Frojmovic Mony
Published: March 2018

Previous studies evaluating requirements for occupancy of thrombin receptors in normal platelet secretion and aggregation, using the thrombin antagonists hirudin and PPACK (D-Phe-Pro-Arg-chloromethylke-tone), have suggested that at low thrombin activating concentrations (0.025–0.13 U/ml), occupancy was required only in the first 45–60 s following activation. In our study, we differentiate between thrombin receptor occupancy requirements for surface expression of secreted adhesive proteins, for activation of GPIIb-IIIa receptors, and for aggregation of washed platelets (WP) in laminar shear flow. Platelets activated with 0.05 U/ml thrombin for 10 min to allow maximal secretion (hereafter referred to as “pre-activated platelets”), then sheared, showed a 50–70% decrease in platelet counts after 60 s of shear. Treatment of pre-activated platelets with hirudin or PPACK produced a 65% reduction of capture efficiencies, αG (reflecting experimental/theoretical initial rates of aggregation), as well as a 30–40% decrease in the surface expression of von Willebrand factor (vWF) and thrombospondin (TSP). However, α-granule membrane P-selectin expression and numbers of activated GPIIb-IIIa receptors were comparable for treated and non-treated platelets. No significant difference in any of the parameters tested was observed when platelets were similarly pre-activated with 0.2 U/ml thrombin, due to treatment with thrombin antagonists. Binding of soluble FITC-vWF (GRGDSP-sensitive) to pre-activated, thrombin antagonist treated platelets, was greatly reduced (≥80%). Soluble Fg was shown to bind to antagonist-treated pre-activated platelets, but could not significantly enhance platelet aggregation. Although occupancy of thrombin receptors by catalytically active thrombin is required transiently for secretion and activation of platelets, there is a further requirement for thrombin occupancy at low thrombin concentrations, for optimizing initial rates of platelet aggregation, surface expression of vWF and TSP, and activated GPIIb-IIIa ligand recognition.
Recombinant vWF Type 2A Mutants R834Q and R834W Show a Defect in Mediating Platelet Adhesion to Collagen, Independent of Enhanced Sensitivity to a Plasma Protease
Authors: Lankhof Hanneke , Damas Conchi , Schiphorst Marion , IJsseldijk Martin , Bracke Madelon , Furlan Miha , de Groot Philip , Vink Tom , Sixma Jan
Published: March 2018

Type 2A von Willebrand Disease (vWD) is characterized by the absence of high molecular weight von Willebrand factor (vWF) multimers in plasma which is caused by enhanced extracellular proteolysis or defective intracellular transport. We identified in vWD type 2A patients two mutations in the A2 domain at position 834 in which arginine (R) was substituted for glutamine (R834Q) or tryptophan (R834W). We reproduced these mutations in vWF cDNA and expressed the recombinant proteins in furin cDNA containing baby hamster kidney (fur-BHK) cells. The subunit composition and the multimeric structure of both mutants was similar to wild-type (WT) vWF. Characterization of mutant R834Q by ristocetin or botrocetin induced platelet binding, and by binding to heparin showed no abnormality. R834W had normal botrocetin induced platelet binding, but ristocetin induced platelet binding and binding to heparin were decreased. Under static conditions R834Q and R834W, at 10 μg/ml, bound equally well to collagen type III as WT-vWF. At high shear rate conditions both mutants supported platelet adhesion normally when coated to a glass surface or preincubated on collagen. When R834Q or R834W was added to the perfusate, adhesion to collagen type III was 50% of the WT-vWF value, which was not due to a decreased collagen binding under flow. A divalent cation dependent protease, purified from plasma, degraded the 2A mutants rapidly while WT-vWF was not affected. In conclusion, the mutations present in the A2 domain of vWF result in an enhanced proteolytic sensitivity to a divalent ion-dependent protease. When present in the perfusate, R834Q and R834W show a decrease in platelet adhesion to collagen type III under flow conditions, which is not caused by decreased binding of the mutant vWF to collagen or enhanced proteolysis.
Stimulation of Proteinase Activated Receptor-2 Causes Endothelial Cells to Promote Blood Coagulation In Vitro
Authors: Norström Eva , Sundelin Johan , Nystedt Sverker , Alm Anna-Karin
Published: March 2018

Proteolytically activated receptors define a new subclass among the G-protein coupled receptors. Proteinase activated receptor-2 (PAR-2), the second member to be identified of this growing receptor subclass, can be activated by trypsin and trypsin-like serine proteases such as mast cell tryptase. PAR-2 is expressed in endothelial cells. Here we have studied if activation of PAR-2 changes the coagulation properties of cultured human umbilical vein endothelial cells. We show that activation of PAR-2 induces rapid and transient formation of tissue factor mRNA with a maximum level 1 hour after receptor stimulation. The increased mRNA level was accompanied by an increased tissue factor activity at the endothelial cell surface, shortening coagulation time in a standard clotting assay. The level of tissue factor activity after PAR-2 activation was comparable with the effects of thrombin receptor (PAR-1) activation although neither of the two protease receptors were as strong inducers of tissue factor as tumor necrosis factor-α.
Geographic Distribution of the 20210 G to A Prothrombin Variant
Authors: Doggen C. J. M. , Zivelin A. , Arruda V. R. , Aiach M. , Siscovick D. S. , Hillarp A. , Watzke H. H. , Bernardi F. , Cumming A. M. , Preston F. E. , Reitsma P. H. , Rosendaal F. R.
Published: March 2018

A variant in prothrombin (clotting factor II), a G to A transition at nucleotide position 20210, has recently been shown to be associated with the prothrombin plasma levels and the risk of both venous and arterial thrombosis. The purpose of this study was to investigate the prevalence of carriership of this mutation in various populations.

We combined data from 11 centres in nine countries, where tests for this mutation had been performed in groups representing the general population. We calculated an overall prevalence estimate, by a precision-weighted method, and, since the distribution of the prevalences did not appear homogeneous, by an unweighted average of the prevalences. We examined differences in the prevalences by geographical location and ethnic background as a possible explanation for the heterogeneity.

Among a total of 5527 individuals who had been tested, 111 heterozygous carriers of the 20210A mutation were found. The prevalence estimates varied from 0.7 to 4.0 between the centres. The overall prevalence estimate was 2.0 percent (CI95 1.4-2.6%). The variation around the summary estimate appeared more than was expected by chance alone, and this heterogeneity could be explained by geographic differences. In southern Europe, the prevalence was 3.0 percent (CI95 2.3 to 3.7%), nearly twice as high as the prevalence in northern Europe (1.7%, CI95 1.3 to 2.2%). The prothrombin variant appeared very rare in individuals from Asian and African descent.

The 20210A prothrombin variant is a common abnormality, with a prevalence of carriership between one and four percent. It is more common in southern than in northern Europe. Since this distribution within Europe is very different to that of another prothrombotic mutation (factor V Leiden or factor V R506Q), founder effects are the most likely explanation for the geographical distribution of both mutations.
Anti-hepatitis G E2 Antibody Detection and Its Relation to Serum HGV-RNA in Patients with Clotting Disorders: High Prevalence of HGV Infection and Spontaneous Remission
Authors: Sheng L. , Soumillion A. , Peerlinck K. , Verslype C. , Schelstraete R. , Gyselinck F. , Emonds M.-P. , Hess G. , Vermylen J. , Desmyter J. , Yap S. H.
Published: March 2018

In a previous study, we have determined the prevalence of serum HGV-RNA in patients with congenital clotting disorders. Twenty-six (15%) of 175 patients investigated were serum HGV-RNA positive. In addition, HGV-RNA was detectable in peripheral blood mononuclear cells (PBMC) in ten percent of the cases, three of these patients were serum HGV-RNA negative.

In the present study, we have determined the prevalence of anti-HGV-E2 antibodies in the same patient population. Anti-HGV-E2 as determined by ELISA was detected in 45 patients (25.7%). Forty of these patients were serum HGV-RNA negative.

Ninety-two percent of the 26 HGV viremic patients and all but one patient (44 patients) with detectable anti-HGV-E2 had coinfection with the hepatitis C virus (HCV). Of these coinfected patients, 62.5% of HGV viremic patients and 53% of anti-HGV-E2 positive patients showed elevated serum ALT levels. Anti-HGV-E2 seroconversion is thus not associated with HCV infection. Two patients who were solely infected with HGV had normal serum ALT levels. In a retrospective longitudinal study, we have observed in 15 patients that serum HGV-RNA persisted during one to 19 years of follow-up, while anti-HGV-E2 was repeatedly negative. Five additional patients who were anti-HGV-E2 positive with concomitant detectable HGV-RNA (4 patients in serum and 1 patient in PBMC) became HGV-RNA negative during follow-up, ranging from 1 to 8 years after the first detection of anti-HGV-E2 antibodies. Two patients had lost anti-HGV-E2 antibodies 3 to 6 years after the seroconversion without the re-appearance of serum HGV-RNA. From these findings, it is clear that the prevalence rate of HGV infection in patients with clotting disorders as determined by PCR assay for HGV-RNA and anti-HGV-E2 by ELISA is actually higher than the prevalence of HGV viremia. Although HGV viremia may persist for longer than 19 years, most of the patients infected with HGV may clear the viremia spontaneously. The clearance of viremia is usually associated with seroconversion to anti-HGVE2. In addition, anti-HGV-E2 may be lost during years of follow-up without the reappearance of the HGV-RNA. Although HGV infection does not seem to influence the fate of HCV infection and does not induce increased levels of serum ALT, the clinical significance of long-term infection remains to be established.
Homozygous Protein C Deficiency: Description of a New Mutation and Successful Treatment with Low Molecular Weight Heparin
Authors: Monagle Paul , Andrew Maureen , Halton Jacqueline , Marlar Richard , Jardine Lawrence , Vegh Patricia , Johnston Marilyn , Webber Colin , Massicotte Patricia
Published: March 2018

We present a kindred with a new mutation of the protein C gene, in which the proband had an unusual clinical presentation. The relationship between warfarin induced skin necrosis and level of anticoagulation was investigated. The pharmacokinetics of protein C concentrate was assessed to determine frequency of replacement therapy. The clinical and biochemical efficacy of therapy with low molecular weight heparin (LMWH) was assessed. The effect of long-term LMWH on bone density in the growing child was monitored using whole body densitometry.

Warfarin therapy required an INR of greater than 3.5 to avoid skin necrosis. If protein C replacement was to be used, doses of 100 U/kg/day would have been required to maintain protein C levels consistently at or above 0.20 U/ml. While receiving prophylactic therapy with LMWH for almost 3 years, there were no episodes of recurrent thrombosis, no skin necrosis and no bleeding. Biochemical markers of in vivo thrombin generation were suppressed and within the normal range. Bone density continued to increase at the normal rate throughout the treatment period.

LMWH is an effective form of long-term therapy for homozygous protein C deficient patients with measurable protein C levels.
Fibrinogen Coating Density Affects the Conformation of Immobilized Fibrinogen: Implications for Platelet Adhesion and Spreading
Authors: Moskowitz Keith , Kudryk Bohdan , Coller Barry
Published: March 2018

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/ IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 μg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 μg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for γ 1-78 and 95-265, respectively, suggesting that the fibrinogen γ-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 μg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at >30 μg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.
Adenosine 3’:5’-Cyclic Monophosphate Induces Regulated Secretion of Tissue-Type Plasminogen Activator and von Willebrand Factor from Cultured Human Endothelial Cells
Authors: Hegeman R. J. , Eijnden-Schrauwen van den , Emeis J. J.
Published: March 2018

The effect of compounds increasing intracellular adenosine 3’:5’-cyclic monophosphate [cAMP]i levels (prostacyclin, isoproterenol, forskolin, cholera toxin), and of the cAMP analogs 8-bromo-cAMP and dibutyryl-cAMP, on the regulated secretion (acute release) of tissue-type plasminogen activator (tPA) and von Willebrand factor (vWF) was studied in cultured human umbilical vein endothelial cells (HUVEC).

Prostacyclin, isoproterenol and forskolin, which increased [cAMP]i in HUVEC, and the cell-permeant cAMP analog 8-bromo-cAMP induced dose- and time-dependent secretion of tPA and vWF. The extent of vWF and tPA release correlated with [cAMP]i, and was increased by the phosphodiesterase inhibitor isobutylmethylxanthine.

In contrast to thrombin, the cAMP-elevating agents did not increase the intracellular calcium concentration [Ca2+]i in HUVEC. At sub-maximal concentrations, the effects of thrombin and prostacyclin were additive.

Our results show that an increase in [cAMP]i resulted in regulated secretion (acute release) of tPA and vWF from HUVEC, without the concomitant increase in [Ca2+]i which is, in HUVEC, essential for thrombin-induced regulated secretion to occur. cAMP-induced secretion represents a novel mechanism for causing regulated secretion of tPA and vWF from endothelial cells.
A Founder Effect Is Proposed for Factor XIII B Subunit Deficiency Caused by the Insertion of Triplet AAC in Exon III Encoding the Second Sushi Domain
Authors: Souri Masayoshi , Izumi Tomonori , Higashi Yoichiro , Girolami Antonio , Ichinose Akitada
Published: March 2018

We previously concluded that genetic defects in the B subunit of factor XIII were the basis for former Type I deficiency (i.e. factor XIII B subunit deficiency). When we examined an Italian patient with the disease at the DNA level, restriction digestion and sequencing analyses of amplified DNAs revealed that the proband and her family members possessed an AAC insertion within the codon for Tyr-80 in exon III in the gene for the B subunit, a nucleotide polymorphism (A-G) in its 3’-noncoding region in exon XII, and a short tandem repeat polymorphism of (TTTA)9 in the 3’-flanking region. These mutations and 3’-polymorphisms were also identified in another Italian family reported in a previous study (10), suggesting that a founder effect is responsible for factor XIII B subunit deficiency in Italians.
Coagulation Factor V (Arg506 → Gln) Mutation and Early Saphenous Vein Graft Occlusion after Coronary Artery Bypass Grafting
Authors: Silveira Angela , Ferdinand van’t Hooft , Tornvall Per , Blombäck Margareta , Wiman Björn , Rydén Lars , Hamsten Anders , Moor Elisabeth
Published: March 2018

The factor V (Arg506→Gln) mutation confers an increased risk of deep vein thrombosis, whereas its role in saphenous vein graft closure after coronary artery bypass grafting (CABG) remains unclear. This study examined the anticoagulant response to activated protein C (APC ratio) in relation to the surgical trauma and the significance of the factor V Leiden mutation in determining postoperative thrombin generation and fibrin formation and the risk of early vein graft occlusion. A total of 108 men undergoing elective CABG for exertional angina pectoris (mean age 61.1 ± 8.7 years) were examined. The patency of saphenous vein grafts was studied at routine reangiography three months after CABG.

Of 100 patients who underwent reangiography, 23 had one or more occluded vein grafts at reangiography. Heterozygosity for the factor V (Arg506→Gln) mutation tended to be associated with early saphenous vein graft occlusion (5/11 carriers vs. 18/89 non-carriers with graft occlusion, व2 = 3.52, p = 0.06), whereas pre- and postoperative APC ratios did not. Pre- and postoperative determinations of prothrombin fragment 1+2, thrombin-antithrombin complexes and soluble fibrin levels did not differ between patients with and without the mutation.

Early saphenous vein graft occlusion after CABG could tentatively be added to deep vein thrombosis as a vascular complication that can be attributed to the factor V (Arg506→Gln) mutation.
Congenital Resistance to Activated Protein C in Patients with Lupus Anticoagulants: Evaluation of Two Functional Assays
Authors: Duca Francesca , Ruggeri Luisa , Finazzi Guido , Negri Barbara , Moia Marco , Galli Monica
Published: March 2018

The R506Q mutation (“Factor V Leiden”) is responsible for the resistance to activated Protein C (aPCR), that is evaluated by coagulation tests. Such tests cannot be used in patients with lupus anticoagulants (LAs), due to the interfering effect exerted by these antibodies on “in vitro” phospholipid-dependent coagulation tests. For this reason, assays have been developed to evaluate aPCR that are insensitive to the presence of LA antibodies. We evaluated two such coagulation tests in the plasma of 82 consecutive patients with LAs. By polymerase chain reaction 3 patients (3.6%) were found heterozygous for the R506Q mutation. aPCR was evaluated by two clotting assays, proposed to be “insensitive” to the presence of LAs: 1. aPCR-tissue factor-based assay, using Factor V deficient plasma and 1:40 diluted test plasma; 2. aPCR-dRVVT-based assay with highly concentrated phospholipids. Their interassay coefficient of variation was 28% and 6.2%, respectively. Compared to the polymerase chain reaction analysis, the 2 tests displayed the following characteristics: sensitivity 67% vs 100%, specificity 92% vs 96%, positive predictive value 25% vs 50%, negative predictive value 99% vs 100%, respectively. Among LA patients without the R506Q mutation, 5 scored positive in the aPCR-tissue factor-based assay, 2 in the aPCR-dRVVT-based assay and another one in both assays. Our findings suggest that the aPCRdRVVT-based test is more reliable and sensitive than the aPCR-tissue factor-based one to the R506Q mutation in patients with LAs. Both assays, when negative, make unlikely the presence of the R506Q mutation. Polymerase chain reaction analysis remains, however, to be performed when either test is positive.
Assessment of the Influence of Citrate Concentration on the International Normalized Ratio (INR) Determined with Twelve Reagent-instrument Combinations
Authors: Chantarangkul Veena , Clerici Marigrazia , Negri Barbara , Mannucci Pier Mannuccio , Tripodi Armando
Published: March 2018

Citrate concentration is one of the variables that can affect coagulation tests. However, few studies have so far been performed to assess the magnitude of this effect on coagulation tests in general and PT in particular. The aim of this study was to assess the extent of influence of citrate concentration on the PT test with results expressed as INR. Twelve reagent-instrument combinations (systems) were calibrated vs. the Reference Preparation BCT/441 using plasmas collected in either 105 mM or 129 mM citrate from normals and anticoagulated patients (OAT). PTs for plasmas collected in 129 mM citrate were longer than those collected in 105 mM both for normals and patients on OAT, but the ratios (patient-to-normal clotting times) for the two citrate concentrations were significantly different in many instances, implying that the International Sensitivity Index (ISI) is also different. ISIs for calibrations with plasmas collected in 105 mM were greater (up to 10%) than those with plasmas collected in 129 mM citrate. When PT ratios were transformed into INR using crossover ISIs (i.e., plasmas collected in 105 mM and ISI determined with plasmas collected in 129 mM citrate, or vice versa) we found that an INR of 4.5 could be up to 20% apart from the value that would have been obtained if the appropriate ISI was used. Moreover, if the ISI determined with the manual technique was used to convert PTs obtained with a particular instrument into INR, the effect of citrate concentration was even greater (INR difference up to 64%). Should these observations be valid for other systems, they might provide additional explanations for the frequent reports which document discrepancies in the INR determined with different systems to which incorrect ISI might have been applied. World-wide consensus on a single citrate concentration to collect patients’ as well as lyophilized plasmas to be used in External Quality Assessment Schemes and for local system calibration is therefore urgently needed.