bluuebunny/crossref_metadata_2025 huggingface dataset:
Ecarin Clotting Time: a Predictive Coagulation Assay for the Antithrombotic Activity of Argatroban in the Rat
Authors: Lunven Catherine , Girardot Christine , Lechaire Irène , Girard Denise , Charles Marie-Christine , Ferrari Patrice , O’Brien Donogh , Berry Christopher
Published: March 2018

We studied the use of the Ecarin Clotting Time (ECT) as a predictive assay of the antithrombotic effects of argatroban in a new tissue factor-dependent model of venous thrombosis and a model of arterial thrombosis in the rat. Heparin was used as a reference anticoagulant.

Infusions of argatroban dose-dependently increased the ECT across the range of doses required for antithrombotic activity in models of venous and arterial thrombosis (1.25-40 μg/kg/min). The TT was only useful as a marker in the case of venous thrombosis, since, in the arterial thrombosis model, the clotting times were >200 s in the majority of animals receiving antithrombotic doses. The aPTT is not sufficiently sensitive to be predictive of an antithrombotic effect in the venous model, and shows only modest increases in the arterial thrombosis model. Heparin did not significantly increase the ECT at antithrombotic doses in the venous thrombosis model, and only increased the ECT by 53% at 40 μg/kg/min in the arterial model, despite a marked antithrombotic effect. Both the TT and aPTT were dose-dependently increased by heparin at doses active in the venous model, whereas both parameters were >200 s at doses active in the arterial thrombosis model.

Thus, the ECT provides a predictive marker for the antithrombotic activity of argatroban in both venous and arterial thrombosis, at least in the rat.
Expression of Thrombospondin 1 on the Surface of Activated Platelets Mediates their Interaction with the Heavy Chains of Human Kininogens through Lys 244-Pro 254
Authors: Kunapuli Satya , Walz Daniel , Colman Robert , DeLa Cadena Raul
Published: March 2018

Platelet thrombospondin (TSP1) forms a complex with high (HK) and low (LK) molecular weight kininogens. We isolated a proteolytic fragment from HK and LK heavy chains (12 kDa) recognized by TSP1 with a N-terminal sequence, K244ICVGCPRDIP254. Lys244-Pro254 oxidized to cyclic form prevented binding of 125I-LK to TSP1. This effect was abolished by reduction and alkylation. Oxidized peptide KICVGCPRDIP (100 μM) reversed the known inhibitory effects of LK or HK (1 μM), on thrombin-induced platelet activation, suggesting this peptide forms part of the cell binding site on HK and LK for activated platelets. KICVGCPRDIP completely inhibited the binding of 125I-LK to activated platelets. However, the peptide only partially inhibited binding of 125I-HK to platelets, suggesting an additional binding site on the HK light chain. Fluorescein-labeled KICVGCPRDIP bound directly and specifically to activated platelets. A monoclonal antibody directed to TSP1 partially inhibited the binding of 125I-HK to activated but not inactivated platelets. We conclude residues Lys244-Pro254 on kininogen heavy chain is responsible for binding to thrombospondin on the surface of activated platelets.
The Effects of Low-intensity Warfarin on Coagulation Activation in Patients with Antiphospholipid Antibodies and Systemic Lupus Erythematosus
Authors: Crowther Mark , Julian James , Stewart Katheryne , Donovan Dianne , Kaminska Elzbieta , Laskin Carl , Ginsberg Jeffrey , Douketis James
Published: March 2018

The optimal intensity of oral anticoagulant therapy for the prevention of thromboembolism in patients with antiphospholipid antibodies (APLA) and systemic lupus erythematosus is controversial. Retrospective studies have suggested that patients with APLA are resistant to oral anticoagulant therapy, with a targeted International Normalization Ratio (INR) of 2.0 to 3.0, and that a higher intensity of anticoagulation (INR: 2.6 to 4.5) is required to prevent recurrent thromboembolism. To investigate if patients with APLA are resistant to the anticoagulant effect of low intensities of warfarin therapy, we performed a randomized trial in which 21 patients with APLA and systemic lupus erythematosus were allocated to receive one of three intensities of warfarin (INR: 1.1 to 1.4, 1.5 to 1.9 or 2.0 to 2.5) or placebo for four months. The main outcome was the effect of each intensity of warfarin therapy on prothrombin fragment 1+2 level (F1+2), that was used as a marker of coagulation activation. When F1+2 levels in patients allocated to the three warfarin intensities were compared to F1+2 levels in the placebo group, there was a statistically significant decrease (p <0.05) in the patient group receiving warfarin with a targeted INR of 2.0 to 2.5 at two, three and four months, and in the patient group with a targeted of INR 1.5 to 1.9 at three months. We conclude that in patients with APLA and systemic lupus erythematosus, warfarin therapy, with a targeted INR of 2.0 to 2.5, is effective in suppressing coagulation activation, and therefore, might be effective in preventing thromboembolism.
Surface-dependent Conformations of Human Fibrinogen Observed by Atomic Force Microscopy under Aqueous Conditions
Authors: Sit P. Sidney , Marchant Roger
Published: March 2018

Conformational differences in human fibrinogen under aqueous conditions on hydrophobic, positively charged and negatively charged surfaces, were examined by atomic force microscopy (AFM). Hydrophobic and positively charged surfaces were prepared by depositing octadecyltrichlorosilane (OTS) and 3-aminopropyltriethoxysilane (APTES) respectively on cleaned glass coverslips forming self-assembled monolayers. The negatively charged surface was prepared by freshly cleaving muscovite mica. AFM operated in fluid tapping mode with an ultrasharp carbon spike probe was used to obtain the molecular scale images. Fibrinogen displayed a characteristic trinodular structure on all three surfaces, although additional U-shaped conformations were observed on mica. In its native hydrated state, fibrinogen is well represented by three connected ellipsoids in close proximity. Quantitative dimensional analysis, which yielded structural information in three dimensions, indicates that surface-dependent structural deformation or spreading of fibrinogen increases according to the order: mica < APTES < OTS. Molecular length, and D and E domain widths of fibrinogen are increased, while the corresponding heights are decreased. The results provide direct evidence that material surface properties affect the conformational state of interacting fibrinogen.
Platelet-vessel Wall Interactions with Third-generation Oral Contraceptives: No Evidence of Detrimental Effects
Authors: Fruzzetti Franca , Giannessi Daniela , Ricci Cabiria , Bernini Walter , Puntoni Roberto , Genazzani Andrea Riccardo , De Caterina Raffaele
Published: March 2018

Because of the association of oral contraceptives (OC) and cigarette smoking with an increased thrombotic risk, we evaluated thromboxane (TX) and prostacyclin urinary (u) metabolites, as in vivo indices of platelet-vessel wall interactions, in women assigned to third generation OC. Twenty-eight women (15 smokers) underwent a 6-month trial of 30 μg ethinylestradiol plus 0.150 mg desogestrel. Cotinine plasma levels were elevated only in persons classified as smokers and serum TXB2 determination confirmed the absence of cyclooxygenase inhibition throughout the study. u-TXB2 and 11-dehydro-TXB2 were higher in smokers than in non-smokers. OC decreased u-11-dehydro-TXB2 both in smokers (from (pg/μmol creatinine) 35.1 ± 6.9 to 15.8 ± 2.8; P <0.025) and non-smokers (from 31.7 ± 9.8 to 20.6 ± 4.8, P = N.S.). u-6-keto-prostaglandin(PG)F1α excretion, also higher in smokers compared to non-smokers, was also reduced after OC in smokers (from (pg/μmol creatinine) 24.3 ± 5.2 to 14.8 ± 2.3; P <0.05). Smokers also had a trend to higher u-2,3-dinor-6-keto-PGF1α, marginally reduced by OC.

Thus, the OC regimen used here improves – if anything – platelet vessel wall interactions as assessed by prostanoid production in vivo. The prothrombotic tendency associated with the use of OC in smokers does not appear to be mediated by changes in platelet-vessel wall interactions.
The Factor II G20210A and Factor V G1691A Gene Transitions and Coronary Heart Disease
Authors: Arsic Tanja , Katz Norbert , Tillmanns Harald , Hehrlein Friedrich , Haberbosch Werner , Gardemann Andreas
Published: March 2018

Background: G to A transitions at nucleotide position 20210 of the factor II (FII) gene and at 1691 of the factor V (FV) gene have been shown to be associated with an increased risk of venous thrombosis. Since it is still unclear whether both gene variations are also related to an increased risk of coronary heart disease (CHD), we studied the relation of both gene variations to coronary artery disease (CAD) and myocardial infarction (MI) in a sample of 2210 male individuals whose coronary anatomy were defined by coronary angiography.

Results: In the total sample, the FII G20210A gene variation was not associated with the presence or the extent of CAD, the latter defined either by the degree of vessel disease or by a CHD score according to Gensini. However, individuals with unfavourable lipid profiles showed pronounced differences in CHD scores between GA heterozygotes and GG homozygotes; this observation applied in particular to younger patients (<62 years; mean age of total sample) who simultaneously had low apoAI/apoB ratios (< 1.19, mean value) and high Lp(a) plasma levels (>28 mg/dl; mean value). In addition, in subjects without acetylsalicylic acid treatment GA heterozygotes had clearly higher CHD scores than AA genotypes. Further restriction to smokers, to subjects with high fibrinogen plasma levels (>3.47 g/l; mean value) or to patients with high glucose concentrations (>112 mg/dl; mean value) tended to increase the difference in CHD score between FII G20210A genotypes. An association of the FII G20210A gene variation with nonfatal MI was not observed. In the total sample and in high and low risk subpopulations, an association of the FV G1691A gene variation was not detected neither with presence and extent of CAD or with nonfatal MI.

Conclusion: The importance of the factor II G20210A gene variation for CHD may be restricted to individuals with major cardiovascular risk factors. In addition, the present study did not strengthen the hypothesis of the factor V G1691A transition as a risk factor of coronary heart disease neither in the total sample nor in subgroups of individuals who were at high or low risk of CHD.
Performances of the Fibrin Monomer Test for the Exclusion of Pulmonary Embolism in Symptomatic Outpatients
Authors: Bounameaux Henri , Perrier Arnaud , Moerloose Philippe , Reber Guido
Published: March 2018

Many studies have shown that D-dimer determinations can be used for the exclusion of venous thromboembolism in symptomatic outpatients, depending however on the method of D-dimer measurement. Another related assay, the Fibrin Monomer test which measures soluble fibrin levels in plasma by ELISA, is now available.

We have evaluated the performances of this assay for the exclusion of pulmonary embolism (PE) in 426 consecutive outpatients presenting at the emergency ward of our institution. Diagnosis of PE was made by D-dimer measurement, compression ultrasonography, lung scintigraphy, venography and pulmonary angiography. With a cut-off of 3 μg/ml, the sensitivity and the negative predictive value were both 100% (95% CI: 97.1-100 and 96.3-100 respectively) and the specificity 33% (95% CI: 25.7-38.1). With 4 μg/ml, the corresponding figures were 98.4 (95% CI: 94.4-99.8), 98.3 (95% CI: 94.1-99.8) and 39% (95% CI: 33.6-44.7) respectively. The prevalence of PE was 30%, the exclusion rates were 23 and 27% for either cut-off. When compared with a reference D-dimer assay (Asserachrom D-Di), a good correlation was observed. In conclusion, this is the first study suggesting the interest of this Fibrin Monomer test to rule out PE; these results, however, need to be confirmed by other studies.
Antithrombotic and Anticoagulant Activities of a Low Molecular Weight Fucoidan by the Subcutaneous Route
Authors: Jouault S. Colliec , Mauray S. , Theveniaux J. , Sternberg C. , Vidal Boisson , Fischer A. M. , Millet J.
Published: March 2018

Fucoidans (high-molecular-weight sulfated polysaccharides extracted from brown seaweeds) have anticoagulant and antithrombotic effects. They inhibit thrombin by catalyzing both serpins (antithrombin and heparin cofactor II) according to their chemical structures and origins. In this study, a low-molecular-weight (LMW) fucoidan of 8 kDa was obtained by chemical degradation of a high-molecular-weight fraction. The antithrombotic and anticoagulant activities of this new compound were compared to those of a low-molecular-weight heparin (LMWH), dalteparin, following subcutaneous administration to rabbits. This LMW fucoidan exhibited dose-related venous antithrombotic activity, with an ED80 of about 20 mg/kg, 2 h after a single subcutaneous injection. Its activity was comparable to that of dalteparin (close to 200 anti-Xa IU/kg) and was maximal 30 min after a single subcutaneous injection. The activity remained stable (about 70%) from 1 to 4 h after injection, but disappeared by 8 h. The antithrombotic activity was not associated with either a prolongation of the thrombin clotting time (TCT) or an increase in anti-Xa activity, contrary to dalteparin. A slight prolongation of APTT occurred with both compounds. This venous antithrombotic activity was associated with a decrease in ex vivo thrombin generation and with a significant increase in the lag phase in a thrombin generation test. LMW fucoidan thus has potent antithrombotic activity and a potentially weaker haemorrhagic effect (i.e. a smaller effect on coagulation tests and a smaller prolongation of the bleeding time) than dalteparin.
Production of Recombinant Human Protein C In Vitro and In Vivo by Muscle Cells
Authors: Türkay Ataç , Wang Jian-Min , Kurachi Kotoku
Published: March 2018

Protein C plays a key role in a natural anticoagulation mechanism, and is also implicated in fibrinolytic and anti-inflammatory functions. Here we describe the production of biologically active human protein C by muscle-targeted gene transfer. Human protein C expression vectors were designed and constructed to produce human protein C in skeletal muscle cells. These vectors were tested in transient and stable transfections of SCID mice myoblasts. Stably transfected cells produced as high as 2.27 μg/106 cells/day. Human protein C produced had a relative activity of 92 ± 8% compared to the plasma derived human protein C, and was composed of α and β forms, 69% and 31%, respectively. After implantation of stably transfected myoblasts into the hind limb muscles of SCID mice, systemic stable production of human protein C in a range of 31-116 ng/ml serum was obtained up to at least 2.5 months.

Current address: J. Wang, Department of Hematology, Changai Hospital, 174 Changai Road, Yangpu District, Shangai 200433, China
Plasmodium Falciparum-infected Erythrocytes: A Mutational Analysis of Cytoadherence via Murine Thrombomodulin
Authors: Rabhi-Sabile Samia , Steiner-Mosonyi Marta , Pollefeyt Saskia , Collen Desiré , Pouvelle B. , Gysin Jurg , Boffa Marie-Claire , Conway Edward
Published: March 2018

The pathophysiologic events leading to organ damage in Plasmo-dium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMser-gly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.
DX-9065a, an Orally Active Factor Xa Inhibitor, Does not Facilitate Haemorrhage Induced by Tail Transection or Gastric Ulcer at the Effective Doses in Rat Thrombosis Model
Authors: Tanabe Kiyoshi , Morishima Yoshiyuki , Shibutani Tomoko , Terada Yasuko , Hara Tsuyoshi , Shinohara Yasutaka , Aoyagi Kazuharu , Kondo Takashi , Kunitada Satoshi
Published: March 2018

DX-9065a is an antithrombin III (AT III)-independent and selective inhibitor of activated blood coagulation factor X (FXa). We evaluated the effects of DX-9065a and warfarin on bleeding time and blood loss in rat tail transection model and on blood loss in hydrochloride (HCl)-induced rat gastrointestinal haemorrhage model. The blood loss was determined by measuring the haemoglobin content in saline immersed with transected tail or hematin chloride content in the gaster after HCl administration. DX-9065a or warfarin was administered orally at 1 h or 15-21 h before the haemorrhagic stimuli, respectively. The dose required for 50% inhibition of thrombus formation (ID50) was 21 mg/kg for DX-9065a and 0.75 mg/kg for warfarin in a copper wire-inserted arteriovenous (AV) shunt model. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) significantly prolonged the bleeding time. In rat tail transection model, the blood loss for the control group was 102 ± 41 μl at 20 min after the transection. While warfarin (0.75 mg/kg) facilitated the blood loss about 5 times as much as the control, DX-9065a (10 or 30 mg/kg) did not. In rat gastrointestinal model, the blood loss for the control group was 15.9 ± 5.6 μl at 15 min after HCl administration. In contrast to DX-9065a (10 or 30 mg/kg), warfarin (0.75 mg/kg) increased the blood loss about twice as much as the control. Thus, compared with warfarin, DX-9065a only increased bleeding time or blood loss to a minor extent in the doses tested. These observations suggest that direct inhibition of FXa could be preferable to warfarin in the suppression of thrombosis without haemorrhagic complications.
Procedure-dependence and Tissue Factor-independence of Hypercoagulability during Orthopaedic Surgery
Authors: Gori A. M. , Falciani M. , Cellai A. P. , Aglietti P. , Baldini A. , Vena L. M. , Prisco D. , Abbate R. , Gensini G. F. , Fedi S.
Published: March 2018

The increased risk for deep vein thrombosis (DVT) after orthopaedic surgery has been well documented as well as hypercoagulable state during both total hip arthroplasty (THA) and total knee replacement (TKR). To investigate the influence of the surgical procedure [postero-lateral (PL) or lateral (L) approach for THA, use of tourniquet (TQ) or not use of TQ for TKR] on the hypercoagulability and the role of extrinsic pathway activation and endothelial stimulation during orthopaedic surgery we have examined 40 patients (20 patients undergoing primary THA – 10 with PL approach and 10 with L approach – and 20 patients undergoing TKR – 10 with TQ application and 10 without TQ). Thrombin-antithrombin complexes (TAT), tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM) and von Willebrand factor antigen (vWF:Ag) were analyzed before and during the orthopaedic surgery. During THA, TAT plasma levels increased more markedly in patients assigned to the L than PL approach (p <0.05); during TKR an elevation of TAT of higher degree (p <0.05) was observed when TQ was not applicated. Blood clotting activation was significantly (p <0.001) more relevant during THA than TKR. No changes in TF and vWF:Ag plasma levels were observed in all patients undergoing THA and TKR. TFPI plasma levels significantly (p <0.05) decreased 1 h after the end of the THA in group PL and group L, whereas they remained unaffected in the two groups of patients undergoing TKR. Similarly TM plasma levels significantly decreased during THA, but not during TKR. In conclusion, these results show that: 1) the site of surgical procedures and the type of approach affect the degree of hypercoagulability, 2) the blood clotting activation takes place in the early phases of orthopaedic surgery, without signs of extrinsic pathway and endothelial activation.
A New Type of Ser Substitution for γ Arg-275 in Fibrinogen Kamogawa I Characterized by Impaired Fibrin Assembly
Authors: Mimuro Jun , Kawata Yoichi , Niwa Kazuki , Muramatsu Shin-ichi , Madoiwa Seiji , Takano Hiroshi , Sugo Teruko , Sakata Yoichi , Sugimoto Tomoyoshi , Nose Koichiro , Matsuda Michio
Published: March 2018

A new type of substitution, Arg to Ser at γ275, has been found in a heterozygous dysfibrinogen derived from a 23-year-old woman with no major bleeding or thrombosis. By sequence analyses of the affected γ-chain and its gene, we found a single amino acid substitution of γ Arg-275 to Ser in an aberrant γ (274-302) residue peptide isolated from lysyl endopeptidase-digests of the patient’s fibrinogen. In agreement with this amino acid substitution, we identified a single nucleotide exchange of A for C at position 5728 in the γ-chain gene creating a codon (AGC) encoding Ser instead of the codon (CGC) encoding Arg at position γ 275. Like two other known types of mutants with a His or Cys substitution at this position, the functional abnormality was characterized by delayed fibrin polymerization, most likely due to impaired abutting of two D domains of adjacent fibrin monomers in the same strand of fibrin protofibrils. The structural derangement that affects the D:D association may not be so severe as compared with those of Cys and His mutants, possessing an additional disulfide-linked Cys molecule and an imidazole ring at the mutation site, respectively.
Effect of Clopidogrel on Thrombin Generation in Platelet-Rich Plasma in the Rat
Authors: Hérault J.-P. , Dol F. , Gaich C. , Bernat A. , Herbert J.-M.
Published: March 2018

Clopidogrel (25 mg/kg, p.o.), a potent and selective inhibitor of ADP-induced platelet aggregation, significantly inhibited, in the presence of platelets, ex vivo thrombin generation triggered by low concentrations of tissue factor. Clopidogrel reduced the area under the curve (23%, p <0.05) and the thrombin peak concentration (35%, p <0.05) but did not affect the lag phase of thrombin generation. Under the same experimental conditions, heparin (100 μg/ml) inhibited thrombin generation mostly by delaying and by reducing the burst of thrombin. In a stasis-induced venous thrombosis model in rats under low thrombogenic challenge, clopidogrel inhibited thrombus formation (ED50 = 7.9 ± 1.5 mg/kg, p.o. – n = 10), confirming the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation and venous thrombosis.
Characterization of the Genetic Defects in Recessive Type 1 and Type 3 von Willebrand Disease Patients of Italian Origin
Authors: Castaman Giancarlo , Vos Hans , Bertina Rogier , Rodeghiero Francesco , Eikenboom Jeroen
Published: March 2018

The genetic defects causing recessive type 1 and type 3 von Wille-brand disease (VWD) in eight families from the northern part of Italy have been investigated. Mutations were identified in 14 of the 16 disease-associated von Willebrand factor (VWF) genes. Only one mutation, a stop codon in exon 45, was previously reported. Several new mutations were identified: one cytosine insertion in exon 42, one guanine deletion in exon 28, one probably complete VWF gene deletion, one substitution in the 3’ splice site of intron 13, one possible gene conversion, and three candidate missense mutations. One missense mutation, the substitution of a cysteine in exon 42, was identified in all type 3 VWD patients that were previously characterized as a subgroup with significant increase of factor VIII procoagulant activity after desmopressin infusion. This paper demonstrates again that the molecular defects of quantitative VWD are diverse and located throughout the entire VWF gene.
Polymorphism of Platelet Membrane Glycoprotein IIIa: Human Platelet Antigen 1b (HPA-1b/PlA2) Is an Inherited Risk Factor for Premature Myocardial Infarction in Coronary Artery Disease
Authors: Zotz Rainer , Winkelmann Bernhard , Nauck Markus , Giers Günther , Maruhn-Debowski Beate , März Winfried , Scharf Rüdiger E.
Published: March 2018

Conflicting results of an association between the human platelet antigen 1b (HPA-1b or PlA2) allele and the risk of myocardial infarction and coronary artery disease have been reported. To assess the reason for this discrepancy, we determined the HPA-1 genotype in 298 men who had undergone coronary angiography, including 124 individuals with myocardial infarction, 83 individuals with coronary artery disease but no history of myocardial infarction, and 91 control patients. Among patients with acute or recent onset myocardial infarction (<1 year), the prevalence of HPA-1b was higher than among patients with coronary artery disease but without myocardial infarction (33 percent vs. 14 percent, p = 0.016). In patients under 60 years of age this difference was even more pronounced (45 percent vs. 15 percent, p = 0.003). Unlike conventional risk factors HPA-1b does not represent a risk factor for coronary artery disease itself but appears to be associated with increased platelet thrombogenicity.
Relations of Platelet Ca2+ Handling and Membrane Microviscosity to Vascular Tone and Restenosis after Angioplasty in Human Coronary Artery
Authors: Feuvre Claude Le , Brunet Annie , Catuli Didier , Metzger Jean-Philippe , Vacheron André , Devynck Marie-Aude , Sang Kim-Hanh Le Quan
Published: March 2018

This study was designed to assess whether platelet Ca2+ handling or membrane microviscosity could be considered as indexes of vascular tone, or could help to predict an increased risk of restenosis after coronary angioplasty. Vascular tone was quantified in 21 patients with stable angina by the vasodilator response to sin-1 intracoronary injection in the reference coronary segment and by the importance of the acute recoil after angioplasty in the narrowed segment. The degree of restenosis was quantified by coronary angiography 6 months later.

Individual values of relative sin-1-induced changes in the reference coronary diameter were positively correlated with cytosolic Ca2+ concentration in unstimulated platelets, irrespective of the extracellular Ca2+ concentration (p <0.01). This relationship was also observed with the thrombin-evoked Ca2+ changes, measured in the absence of a Ca2+ influx (p = 0.01). No relationship was found between sin-1-induced coronary changes and membrane microviscosity evaluated by TMADPH and DPH anisotropies or platelet volume, or between degree of acute recoil and platelet characteristics.

In conclusion, platelet Ca2+ reflects the vasodilating efficacy in response to sin-1, but cannot help to predict restenosis after coronary angioplasty.
Effect of SR121566A, a Potent GP IIb-IIIa Antagonist, on the HIT Serum/heparin-Induced Platelet Mediated Activation of Human Endothelial Cells
Authors: Savi Pierre , Jeske Walter , Walenga Jeanine , Herbert Jean-Marc
Published: March 2018

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1β, IL6, TNFα and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-IIIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPγS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPγS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs.

Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.
Hemostatic Effects of Oral Contraceptives in Women who Developed Deep-vein Thrombosis while Using Oral Contraceptives
Authors: Bloemenkamp Kitty , Helmerhorst Frans , Koster Ted , Bertina Rogier , Vandenbroucke Jan , Rosendaal Frits
Published: March 2018

Objective: Comparison of the effect of oral contraceptives on hemostatic variables in venous thrombosis patients (thrombosis while using oral contraceptives) with the effect in healthy control subjects. Our aim was to assess whether some of these effects were more pronounced in women who had suffered thrombosis, i.e., whether these were “hemostatic hyperresponders”. Study Design: A population-based case-control study, the Leiden Thrombophilia Study. Materials and Methods: We investigated 99 pre-menopausal women, age 15-49 years, who had used oral contraceptives at the time of a first, objectively confirmed episode of deep-vein thrombosis. They were not pregnant, nor in puerperium, nor had had a recent miscarriage, and were not using injectable progestogens, nor suffering from inherited coagulation defects. The median time between occurrence of deep-vein thrombosis and venepuncture was 18 months, and 30 of the 99 women were still using oral contraceptives, while 69 had discontinued oral contraceptive use. In addition, a group of 153 control women (54 of them were oral contraceptive users and 99 were non-users) were studied. The following hemostatic variables were measured: APTT, factor VII, factor VIII, factor XII, fibrinogen, prothrombin, total antithrombin, normalised activated protein C sensitivity ratio (n-APC-sr), protein C, protein S and free protein S. Results: We found marked and significant effects of oral contraceptive use on the levels of several clotting factors, with an increase in factor VII, factor XII, protein C and a decrease in antithrombin, n-APC-sr and protein S. Less marked effects that were non-significant or only significant in either patients or controls, were an increase in factor VIII, fibrinogen and prothrombin and a decrease in the APTT and free protein S. In the former thrombosis patients several of these effects of oral contraceptives were more pronounced than in healthy women: specifically on factor VII, antithrombin, n-APC-sr and protein C. Conclusions: Our results of the effects of oral contraceptives generally confirm previous reports in healthy volunteers. Our data also show that in former deep-vein thrombosis patients these effects are more pronounced. Apparently some women become “high hemostatic responders” when exposed to oral contraceptives, and they may be the women most vulnerable to its thrombogenic effects.
Molecular Bases of Pseudo-homozygous APC Resistance: The Compound Heterozygosity for FV R506Q and a FV Null Mutation Results in the Exclusive Presence of FV Leiden Molecules in Plasma
Authors: Castoldi Elisabetta , Kalafatis Michael , Lunghi Barbara , Simioni Paolo , Ioannou Panayiotis , Petio Margherita , Girolami Antonio , Mann Kenneth , Bernardi Francesco
Published: March 2018

Pseudo-homozygous APC resistance, the condition resulting from compound heterozygosity for FV R506Q (FV Leiden) and quantitative FV deficiency, provides a natural model to study the interaction between procoagulant and anticoagulant defects. This paper reports a complete FV characterization of a pseudo-homozygous APC resistant thrombotic patient. The expression of the patient’s non-Leiden gene was found to be severely impaired both at the mRNA and protein levels. In particular, only FV Leiden molecules were detected in the patient’s plasma by immunoblotting, which accounts for the observed marked APC resistance. Analysis of the FV cDNA obtained by reverse transcription of platelet RNA revealed that the mRNA of the non-Leiden gene was extremely reduced in amount. A PAC clone containing the whole FV gene was used to design primers for a complete FV exon scanning. A 2-bp insertion at nucleotide 3706 in the large exon 13 of the non-Leiden gene, predicting a frame-shift and premature termination of protein synthesis, was identified as responsible for the FV defect. Failure to find any case of pseudo-homozygous APC resistance in a large sample (6,804) of blood donors suggests that this condition is extremely rare among normal controls and that its detection is favoured by the thrombotic risk that it may confer.
Thrombolysis in Arterial Occlusion
Authors: Verhaeghe Raymond
Published: March 2018

Intra-arterial thrombolytic therapy has replaced systemic intravenous infusion of thrombolytic agents as a treatment modality for arterial occlusion in the limbs. Several catheter-guided techniques and various infusion methods and schemes have been developed. At present there is no scientific proof of definite superiority of any agent in terms of efficacy or safety but clinical practice favours the use of urokinase or alteplase. Studies which compared thrombolysis to surgical intervention suggest that thrombolytic therapy is an appropriate initial management in patients with acute occlusion of a native leg artery or a bypass graft. Underlying causative lesions are treated in a second step by endovascular or open surgical techniques. Severe bleeding is the most feared complication: the risk of hemorrhagic stroke is 1-2%.
Generation of a Humanized, High Affinity Anti-tissue Factor Antibody for Use as a Novel Antithrombotic Therapeutic
Authors: Sims Paul , Meng Y. Gloria , Moran Paul , Bullens Sherron , Bunting Stuart , Schoenfeld Jill , Lowe David , Lai Jadine , Rancatore Patricia , Iverson Mark , Lim Amy , Chisholm Vanessa , Kelley Robert , Riederer Markus , Kirchhofer Daniel , Presta Leonard
Published: March 2018

Blocking the cofactor function of human tissue factor may be beneficial in various coagulation-mediated diseases. The murine antibody D3 binds to the membrane proximal substrate interaction region of human tissue factor and blocks tissue factor function even in the presence of bound factor VIIa. The cloned murine D3 antibody was humanized and affinity matured by exchanging amino acids in the complementarity determining regions as well as in the antibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (KD 0.1 nM) as compared to the original murine and chimeric versions. Depending on the particular disease, different pharmacokinetic properties of the antibody may be required and, therefore, several antibody variants – F(ab), F(ab’)2, IgG2, IgG4 and IgG4b – were generated. In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor VIIa-mediated activation of factors IX and X (e. g. D3H44-F(ab’)2, IC50 (F.X) 47 pM). In addition, D3H44-F(ab’)2 completely prevented fibrin deposition in a human ex vivo thrombosis model under venous blood flow conditions (IC50 37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases which involve tissue factor activity, e. g. acute coronary syndrome and venous thrombosis.
Signaling Through Platelet Integrin αIIbβ3: Inside-out, Outside-in, and Sideways
Authors: Shattil Sanford
Published: March 2018

Introduction

Cell adhesion, which plays a major role in the response of tissues to injury, involves a wide variety of cells, adhesion receptors, and ligands. Prominent among the cells responding to injury are platelets, the first line of defense against hemorrhage and an early player in the overall process of wound-healing.1 Prominent among the adhesion receptors is αIIbβ3, a platelet-specific integrin essential for hemostasis by virtue of its role in mediating platelet aggregation and platelet spreading on vascular matrices. Prominent among the ligands for αIIbβ3 are the multivalent adhesive proteins, fibrinogen and von Willebrand factor (vWF), which in soluble form, mediate platelet aggregation and, in solid phase, mediate adhesive spreading.2

In addition to the adhesive functions of αIIbβ3, this integrin serves as a bidirectional conduit for biochemical and mechanical information flow across the platelet plasma membrane.3 For example, intracellular signals modulate the ligand-binding function of αIIbβ3 (inside-out signaling), and signals generated by ligation and clustering of αIIbβ3 regulate the extent of platelet aggregation and spreading (outside-in signaling). The relationships between αIIbβ3 and intracellular signaling pathways have been the subject of intense study in human platelets, model cell systems, and more recently, in genetically-modified mice. Although model cells and mouse platelets have become indispensable tools to dissect mechanisms of αIIbβ3 signaling, conclusions drawn from their use must always be validated in human platelets. Despite this caveat, a broad, but still incomplete, picture of integrin signaling in platelets is beginning to emerge. This update will highlight selected recent developments in this area and discuss potential clinical implications.
Treatment of Patients with Factor VIII and Factor IX Inhibitors with Special Focus on the Use of Recombinant Factor VIIa
Authors: Hedner Ulla
Published: March 2018

Introduction

In caring for patients with hemophilia, the development of inhibitors against the missing coagulation protein remains a significant problem. Such inhibitors occur in about 15% of patients with severe hemophilia1 and are directed against the procoagulant part of the factor VIII or factor IX molecules. The goal in the treatment of patients with hemophilia is to achieve high enough levels of the missing factor to induce hemostasis in case of bleeding or to prevent bleeding during surgery. Patients with a low inhibitor titer, and especially those with a low anamnestic response, can be given high doses of the coagulation factor concentrate to induce hemostasis. However, for patients with high inhibitor levels, or high-responders, other treatments must be used, including procedures to decrease the antibody titers. To minimize the booster effect of high doses of antigen, immunosuppressive treatment may be added. Because such treatment procedures are complicated and associated with a number of potential side effects, they are not often used to treat mild-to-moderate bleeding episodes or to cover elective, less urgent surgery for inhibitor patients. As a result, a great deal of effort has been devoted to finding more convenient treatment modalities and to inducing immunologic tolerance to permanently eradicate the inhibitors.
Regulation of the Procoagulant Response to Arterial Injury
Authors: Giesen Peter , Schecter Alison , Nemerson Yale , Taubman Mark
Published: March 2018

Introduction

Thrombosis is commonly associated with arterial injury. Clinically, this is seen with all interventional procedures designed to treat coronary artery stenosis, such as percutaneous transluminal coronary angioplasty (PTCA),1 directional coronary, 1 atherectomy (DCA)2 and coronary artery stenting.3 Acute thrombosis occasionally results in total occlusion of the vessel lumen. However, more typical is the deposition of smaller nonocclusive mural thrombi.

Thrombosis is also a common feature of many animal models of arterial injury.4-8 These models have provided considerable information about the molecular events associated with injury-induced thrombus formation. Platelet adherence to the injured arterial wall occurs within minutes in all models of arterial injury. In some models, platelet deposition is followed by fibrin deposition, a consequence of activation of the coagulation cascade. The presence and extent of fibrin deposition varies with the degree of injury (superficial or deep), the type of vessel (carotid, femoral, aorta, or coronary), the state of the vessel prior to injury (normal, cholesterol fed, previously injured), and the species. In the pig carotid balloon injury model, endothelial denudation in the absence of medial injury is associated with platelet deposition but no fibrin generation. More severe injury, defined by the presence of a medial tear, results in marked platelet accumulation and fibrin generation, even in the presence of high doses of heparin.4,9 Unlike the porcine model, balloon injury to normal rodent arteries is associated with rapid platelet deposition but does not result in significant fibrin deposition, even in the presence of medial injury.5-8

In contrast to that found using normal arteries, fibrin deposition is seen when previously injured rabbit arteries, possessing a neointima, are subjected to a second injury.5,7,8,10 As determined by scanning and transmission electron microscopy, abundant fibrin formation is detected within 30 minutes of the reinjury. Platelet deposition is also more dense after the second injury and associated with fibrin-platelet microthrombi. We have recently found a similar difference between single and double injury in rat aorta and carotid arteries. These studies suggest that, in contrast to the deposition of platelets, which accompanies any injury to the arterial wall, the deposition of fibrin and the formation of large thrombi are more dependent on the type of injury produced and may be regulated by specific processes occurring in the arterial wall.
Matrix Metalloproteinase System Deficiencies and Matrix Degradation
Authors: Collen D. , Lijnen H.R.
Published: March 2018

Introduction

Matrix metalloproteinases (MMPs) constitute a tightly regulated family of enzymes that degrade most components of the extracellular matrix. They are classified based on substrate specificity. Collagenases (MMP-1, interstitial collagenase, EC 3.4.24.7; MMP-8, neutrophil collagenase, EC 3.4.24.34; MMP-13, collagenase-3) degrade connective tissue collagens. Gelatinases (MMP-2, gelatinase A, EC 3.4.24.24; MMP-9, gelatinase B, EC 3.4.24.35) degrade collagen types IV, V, VII and X, elastin, and denatured collagens. Stromelysins (MMP-3, stromelysin-1, EC 3.4.24.17; MMP-10, stromelysin-2, EC 3.4.24.22) and matrilysin (MMP-7, EC 3.4.24.23) degrade the proteoglycan core proteins, laminin, fibronectin, elastin, gelatin, and nonhelical collagen, whereas stromelysin-3 (MMP-11) does not degrade any of the major extracellular matrix components. Macrophage metalloelastase (MMP-12, EC 3.4.24.65) degrades insoluble elastin, collagen IV, fibronectin, laminin, entactin, and proteoglycans.1-7 About 20 members of the MMP family have been identified, including at least four membrane-type (MT) MMPs.8-13

MMPs are generally secreted as zymogens that are extracellularly activated by organomercurial compounds, several proteinases (including plasmin, trypsin, chymotrypsin, kallikrein, cathepsin G, or neutrophil elastase), oxygen free radicals, or by association with the cell surface.1,5,6 Active MMPs are inhibited by specific tissue inhibitors of MMPs (TIMPs) via formation of a noncovalent stoichiometric complex.5,6 Four members of the TIMP family have been identified, of which TIMP-1, synthesized by most types of connective tissue cells and by macrophages, acts against all members of the collagenase, stromelysin, and gelatinase classes.2,14,15

MMPs are mainly involved in the degradation of extracellular matrix components, but may also play a role in activation and processing of cytokines or growth factors,16 in shedding of membrane-bound receptors,17 in liberation of Fas ligand,18 or in activation of tumor necrosis factor-α.19 Degradation of extracellular matrix is required to allow cell migration and tissue remodeling, which play essential roles in many (patho)physiological processes. The role of the plasminogen/plasmin (fibrinolytic) and MMP systems in neointima formation after vascular injury, atherosclerosis, graft arterial disease, myocardial ischemia, angiogenesis, tumor growth and dissemination, infection, and chronic inflammatory disorders of the kidney, lung, gastrointestinal tract, skin, joints, or cornea has recently been reviewed.20 We will discuss some recent data on interactions between the MMP and plasminogen/plasmin systems and their role in neointima formation and atherosclerotic aneurysm formation.
Hemostatic Factors and Inflammatory Disease
Authors: Degen Jay
Published: March 2018

Introduction

Vascular integrity is preserved by a sophisticated system of circulating and cell-associated hemostatic factors that control local thrombin generation, platelet deposition, and the conversion of soluble fibrinogen to an insoluble fibrin matrix.1,2 However, there is considerable evidence that hemostatic factors play both a wider physiological role than simply controlling blood loss, and a wider pathological role than simply triggering inopportune thrombotic events, such as myocardial infarction and stroke. In tissue repair, a crucial physiological process, fibrin(ogen) is thought to provide a critical provisional matrix on which cells can proliferate, organize, and carry out specialized functions. A variety of cell types specifically bind to and migrate on fibrin(ogen) matrices. These include endothelial cells, macrophages, neutrophils, smooth muscle cells, fibroblasts, and keratinocytes.3-8 Direct binding to fibrin(ogen) through both integrin [e.g., αvβ3, α1β5, αMβ2 (CD11b/CD18, Mac-1)] and non-integrin receptors (e.g., intercellular adhesion molecule (ICAM-1)) appears to contribute to these cell-fibrin interactions.8-11 Fibrin(ogen) degradation products have also been reported to have an impressive array of biological activities, including mitogenic, angiogenic, chemotactic, and immunosuppressive activities.12-14 There are now substantial data indicating that fibrin(ogen) may plays an important role in the inflammatory response15,16 and that it may, in fact, direct leukocyte transendothelial cell migration.11 Similarly, through several G-protein coupled protease-activated receptors on fibroblasts, endothelial cells, leukocytes, smooth muscle cells, and other cell types, thrombin is thought to play an important role in inflammatory and fibroproliferative responses.17 Fibrinolytic factors, such as plasmin(ogen), also appear to be important modulators of inflammation.18 Finally, host fibrinogen, prothrombin, plasminogen, plasminogen activator, and other hemostatic factors appear to be crucial to the pathogenesis and virulence of many bacterial species.19-21 Unfortunately, despite a myriad of provocative observations made using in vitro systems, there is little direct in vivo evidence supporting an important role of fibrin(ogen) or other hemostatic factors in either the inflammatory response or disease progression. Direct and definitive analyses have been hampered by the lack of an experimental means to specifically manipulate the level or structure of selected hemostatic factors in vivo. Fortunately, this experimental roadblock has been effectively removed by the development of gene-targeting and gene transfer technologies in mice (see below).