Thrombosis Research

Editorial Board

2012-05-01

Factor XI: Hemostasis, Thrombosis, and Antithrombosis

Rong He, Dong Chen, Shilin He — 2011-12-26

Abstract: Coagulation factor FXI (FXI), a plasma serine protease zymogen, has important roles in both intrinsic and extrinsic coagulation pathways and bridges the initiation and amplification phases of plasmatic hemostasis. Recent studies have provided new insight into the molecular structure and functional features of FXI and have demonstrated distinct structural and biological differences between activated factor XII (FXIIa)–mediated FXI activation and tissue factor/thrombin–mediated FXI activation. The former is important in thrombosis; the latter is more essential in hemostasis. Activated partial thromboplastin tine (aPTT) artificially reflects FXIIa-initiated intrinsic coagulation pathway in vitro. Conversely, FXIIa-inhibited diluted thromboplastin time assay may reflect tissue factor/thrombin–mediated FXI activation in vivo. Further explication of the genetic mutations of FXI deficiency has improved the understanding of the structure-function relationship of FXI. Besides its procoagulant activity, the antifibrinolytic activity of FXI was well documented in a wealth of literature. Finally, the new emerging concept of inhibiting FXI as a novel antithrombotic approach with an improved benefit-risk ratio has been supported through observations from human FXI deficiency and various animal models. Large- and small-molecule FXI inhibitors have shown promising antithrombotic effects. The present review summarizes the recent advancements in the molecular physiology of FXI and the molecular pathogenesis of FXI deficiency and discusses the evidence and progress of FXI-targeting antithrombotics development.

Animal models of trauma-induced coagulopathy

Daniel Frith, Mitchell J. Cohen, Karim Brohi — 2011-12-26

Abstract: Resurgent study of trauma-induced coagulopathy (TIC) has delivered considerable improvements in survival after injury. Robust, valid and clinically relevant experimental models of TIC are essential to support the evolution of our knowledge and management of this condition. The aims of this study were to identify and analyze contemporary animal models of TIC with regard to their ability to accurately characterize known mechanisms of coagulopathy and/or to test the efficacy of therapeutic agents. A literature review was performed.Structured search of the indexed online database MEDLINE/PubMed in July 2010 identified 43 relevant articles containing 23 distinct animal models of TIC. The main aim of 26 studies was to test a therapeutic and the other 17 were conducted to investigate pathophysiology. A preponderance of porcine models was identified. Three new models demonstrating an endogenous acute traumatic coagulopathy (ATC) have offered new insights into the pathophysiology of TIC.Independent or combined effects of induced hypothermia and metabolic acidosis have been extensively evaluated. Recently, a pig model of TIC has been developed that features all major etiologies of TIC, although not in correct chronological order.This review identifies a general lack of experimental research to keep pace with clinical developments. Tissue injury and hemorrhagic shock are fundamental initiating events that prime the hemostatic system for subsequent iatrogenic insults. New animal models utilizing a variety of species that accurately simulate the natural clinical trajectory of trauma are urgently needed.

Impaired glucose metabolism and type 2 diabetes are associated with hypercoagulability: potential role of central adiposity and low-grade inflammation – The Hoorn Study

2011-08-19

Abstract: Introduction: Type 2 diabetes (DM2) is associated with greater risk for cardiovascular disease (CVD), which may, at least partially, be explained by prothrombotic alterations. We therefore investigated; first, the extent to which individuals with impaired glucose metabolism (IGM) and/or DM2 had greater levels of thrombin generation than those with normal glucose metabolism (NGM); and second, whether any differences were independent of other cardiovascular risk factors, such as smoking, hypertension, dyslipidaemia, (micro)albuminuria, glycemic control and (central) adiposity, and/or were potentially ‘mediated’ by low-grade inflammation (high-sensitivity C-reactive protein (hsCRP)).Materials and methods: We studied 744 individuals from the Hoorn Study (275 NGM, 176 IGM and 293 DM2, mean age 68.6±7.1years). Thrombin generation in platelet-poor plasma was measured using the Calibrated Automated Thrombogram and three parameters were derived: lag time, peak height and endogenous thrombin potential (ETP). Data were analyzed with multiple linear regression analyses.Results: After adjustment for age, sex, prior CVD and smoking status, individuals with IGM or DM2 had a longer lag time [ß=0.14min (95% CI: 0.02; 0.26)], higher peak height [ß=7.29 nM (−1.33; 15.91)] and ETP [ß=35.65nM*min (0.97; 70.34)] than those with NGM. These differences were attenuated to ß=0.06min (−0.07; 0.19), 3.82nM (−5.46; 13.10) and 16.34nM*min (−20.92; 53.59), respectively, when further adjusted for waist circumference and hsCRP.Conclusion: Individuals with IGM or DM2 had up to 4% higher thrombin generation compared with NGM, which may be explained, to a great extent, by the greater levels of central adiposity and related low-grade inflammation characterizing these individuals.

Alterations of profibrinolytic receptor annexin A2 in pre-eclampsia: A possible role in placental thrombin formation

Hong Xin, Yi Zhang, Huilan Wang, Shuhan Sun — 2011-08-25

Abstract: Introduction: Deficient fibrinolytic activity due to alterations of profibrinolytic receptor annexin A2 has been shown to be related to increased thrombosis. We compared the expression of annexin A2 in pre-eclampsia with that in normal pregnancies and investigated its relationship to placental thrombin formation.Materials and methods: Sixty patients with pre-eclampsia and 30 matched normal pregnant controls were included in the study. Expression of annexin A2 mRNA in placental tissues was analyzed using quantitative RT-PCR. Annexin A2 protein in placentas as well as blood was determined by western blot analysis and immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of anti- annexin A2 antibodies in peripheral maternal blood.Results: The expression of annexin A2 mRNA in placentas was significantly decreased in pre-eclampsia compared to normal pregnancies. Levels of annexin A2 protein in placentas as well as in peripheral maternal blood were also significantly reduced in patients with pre-eclampsia compared with healthy pregnant women. High titers of anti- annexin A2 antibodies were more frequently detected in pre-eclampsia group. Decreased levels of annexin A2 expression and the presence of anti-annexin A2 antibodies were associated with increased placental thrombosis.Conclusion: The down regulation of annexin A2 expression and the presence of anti-annexin A2 antibodies were correlated to PE and may increase placental thrombin formation.

Association of blood transfusion and venous thromboembolism after colorectal cancer resection

Eleftherios S. Xenos, H. David Vargas, Daniel L. Davenport — 2011-08-29

Abstract: Introduction: Red blood cell (RBC) transfusion is a common event in the perioperative course of patients undergoing surgery. Transfused blood can disrupt the balance of coagulation factors and modulates the inflammatory cascade. Since inflammation and coagulation are tightly coupled, we postulated that RBC transfusion may be associated with the development of venous thromboembolic phenomena. We queried the American College of Surgeons’ National Surgical Quality Improvement Program (ACS NSQIP) database to examine the relationship between intraoperative blood transfusion and development of venous thromboembolism (VTE) in patients undergoing colorectal resection for cancer.Materials and Methods: We analyzed the data from 2005 to 2009 for patients undergoing colorectal resections for cancer based on the primary procedure CPT-4 code and operative ICD-9 diagnosis code. The primary outcome was 30-day deep vein thrombosis (DVT) and/or pulmonary embolism (PE). Intraoperative transfusion of RBC's was categorized as: none, 1–2 units, 3–5 units and 6 units or more. DVT/PE occurrences were analyzed by multivariable forward stepwise regression (p for entry .10) to identify independent predictors of DVT.Results: The database contained 21943 colorectal cancer resections. The DVT rate was 1.4% (306/21943) and the PE rate was 0.8% (180/21943). Patients were diagnosed with both only 40 times and the combined DVT or PE rate (VTE) was 2.0% (446/21943). After adjusting for age, gender, race, ASA (American Society of Anesthesiologists) class, emergency procedure, operative duration and complexity of the procedure (based on Relative Value Units, RVU's), along with six clinical risk factors, intraoperative blood transfusion was a significant risk factor for the development of VTE and the risk increased with increasing number of units transfused. Preoperative hematocrit did not enter the multivariable model as an independent predictor of VTE, nor did open versus laparoscopic resection or wound class.Conclusion: In this study of 21943 patients undergoing colorectal resection for cancer, blood transfusion is associated with increased risk of VTE. Malignancy and surgery are known prothrombotic stimuli, the subset of patients receiving intraoperative RBC transfusion are even more at risk for VTE, emphasizing the need for sensible use of transfusions and rigorous thromboprophylaxis regimens.

Attitudes towards and practice of venous thromboembolism prevention in general internal medicine wards: A multinational survey from member countries of the European Federation of Internal Medicine

Moshe Vardi, Lorenzo Dagna, Michal Haran, Roger Duckit — 2011-08-22

Abstract: Introduction: Hospitalised patients in Internal Medicine departments are at risk of venous thromboembolism (VTE). Adherence to risk stratification methods is poor. We conducted a survey among Internists from member countries of the European Federation of Internal Medicine (EFIM) to assess current knowledge and attitude towards VTE prevention.Methods: A multinational survey of Internists affiliated to EFIM.Results: 226 physicians from 30 countries were included. Seventy nine percent of the physicians were aware of clinical guidelines to prevent VTE. Most considered their knowledge of the guidelines to be moderate. Many had not updated their knowledge recently. The magnitude of the clinical problem was over- and underestimated by many (12.2% and 40.1%, respectively). Only 46.7% thought their patients were mostly receiving proper prophylaxis. Sixty four percent worked in departments without a formal VTE prophylaxis program. Risk of bleeding, lack of awareness and lack of decision support systems were the three most common reasons for deferring treatment (88.6%, 32.3% and 27.9%, respectively). Most of the participants stated that they strongly believe in VTE prophylaxis as an intervention that prevents morbidity and mortality.Conclusions: Despite general awareness of clinical guidelines, many medical wards do not have formal risk assessment methodologies incorporated into their operative workflow. This gap, as well as fear of complications, may be one of the reasons for the low rates of adherence reported by physicians. We speculate that perhaps current guidelines have not been accepted by Internists due to paucity of well defined and validated risk assessment tools.

Socioeconomic and occupational risk factors for venous thromboembolism in Sweden: A nationwide epidemiological study

Bengt Zöller, Xinjun Li, Jan Sundquist, Kristina Sundquist — 2011-08-26

Abstract: Our aims were to investigate possible associations between hospitalisation for venous thromboembolism (VTE) and socioeconomic and occupational factors. A nationwide database was constructed by linking Swedish census data to the Hospital Discharge Register (1990–2007). Hospital diagnoses of VTE were based on the International Classification of Diseases. Standardised incidence ratios were calculated for different socioeconomic and occupational groups. A total of 43063 individuals aged >20years were hospitalised for VTE. Individuals with >12years of education were at lower risk for VTE. Blue-collar workers, farmers, and non-employed individuals had higher risks for VTE, and white collar workers and professionals lower risks. In males and/or females, risks for VTE were increased for assistant nurses; farmers; miners and quarry workers; mechanics, iron and metalware workers; wood workers; food manufacture workers; packers; loaders and warehouse workers; public safety and protection workers; cooks and stewards; home helpers; building caretakers; and cleaners. Decreased risks were observed for technical, chemical, physical, and biological workers; physicians; dentists; nurses; other health and medical workers; teachers, religious, juridical, and other social science-related workers; artistic workers; clerical workers; sale agents; and fishermen, whalers and sealers. High educational level and several occupations requiring high levels of education were protective against VTE, while the risks for VTE were increased for farmers, blue-collar workers and non-employed individuals. The mechanisms are unknown but it might involve persistent psychosocial stress related to low socioeconomic and occupational status.

Health care expenditures associated with venous thromboembolism among children

Sheree L. Boulet, Djesika Amendah, Scott D. Grosse, W. Craig Hooper — 2011-08-29

Abstract: Introduction: We used health insurance claims data from large samples of Medicaid-enrolled and privately insured children to identify children with venous thromboembolism (VTE) and to assess their use of health services and associated expenditures during 2009.Materials and Methods: Data from the 2009 Thomson Reuters MarketScan® Commercial Database and Multi-State Medicaid database were used to estimate annual expenditures for children 1–17years of age with VTE. Generalized linear models were used to calculate adjusted annual expenditures for Medicaid-enrolled and privately insured children with VTE, controlling for age, sex, type of health plan, VTE classification (deep vein thrombosis and/or pulmonary embolism), and type of VTE event (idiopathic or secondary) and race (Medicaid only) or region (Commercial only).Results: During 2009, Medicaid-enrolled and privately insured children with VTE had an average of 1–2 inpatient admissions and 8–10 non-emergency department visits. Unadjusted mean total expenditures were similar for Medicaid-enrolled and privately insured children with VTE, $105,359 and $87,767, respectively. Adjusted mean expenditures for children with secondary VTE were five times higher than for children with idiopathic VTE.Conclusions: Given the high frequency of secondary VTE in children, most of the associated expenditures may be due to other health conditions. However, children who develop a VTE incur substantial costs of care, even in the absence of related conditions. Additional research is needed to evaluate the long term outcomes for children with VTE including rates of readmission, complications, and the impact of co-morbid conditions.

Risk of bleeding in low-risk atrial fibrillation patients on warfarin waiting for elective cardioversion

2011-10-05

Abstract: Introduction: Systemic embolism is the most serious complication of cardioversion of atrial fibrillation (AF) and the immediate post-cardioversion period is associated with increased risk for thrombus formation. For this reason, treatment with vitamin K antagonist (VKA) is recommended for patients with AF. No information is available about bleeding risk related to this practice.Methods: We performed a prospective multicentre study on 242 low-risk AF patients (CHADS2 score 0–1) that started on warfarin for elective cardioversion to evaluate their bleeding risk.Results: 178 were males (73.6%), mean age 63.9±9.8years, 60 patients (25%) were aged ≤59years. Patients with CHADS2 score=0 were 73 (30%), those with CHADS2 score=1 were 169 (70%). Patients were on VKA treatment, maintained at INR intended therapeutic range 2.0-3.0, for a median time of 159days (range 30–631)total follow-up period 127 patient-years (pt-yrs). Quality of anticoagulation and occurrence of bleeding events were recorded. Patients spent 23%, 64% and 8% of time below, within and above the intended therapeutic range, respectively. When we observed the INR levels, we found that 62 patients (25.6%) had INR>4.5 at least in one occasion, and 23 (9.5%) in ≥2. During follow-up, 2 patients had major bleeds (rate 1.6% pt-yrs), one fatal. No embolic complications were recorded.Conclusion: Our results show that low-risk AF patients, treated with VKA for elective cardioversion, carry a not irrelevant risk of bleeding. Efforts are required to properly select patients who could benefit from this procedure, reducing the time of warfarin exposure.

Circulating microparticles and risk of venous thromboembolism

2011-09-12

Abstract: Introduction: Circulating microparticles (MPs) may trigger a hypercoagulable state, leading to thrombotic complications. Data on their association with venous thromboembolism (VTE) are few and inconsistent.Materials and methods: To investigate whether or not high levels of MPs are associated with an increased risk of VTE, we carried out a case-control study on 186 patients with a first, objectively diagnosed, episode of VTE and 418 healthy controls. Plasma levels of circulating MPs were measured by flow cytometry.Results: Patients had higher median plasma levels of total MPs than controls (2184 per μL vs 1769 per μL, p<0.0001). The risk of VTE increased progressively with increasing MPs, with a linear dose-response effect in the log odds. Individuals with MPs above the 90th percentile of the controls’ distribution (P90=3263 per μL) had a 5-fold increased risk of VTE than those with MPs below the 10th percentile of controls (P10=913 per μL), independently of sex, age, body mass index, thrombophilia, and plasma factor VIII levels [adjusted odds ratio: 5.30 (95%CI: 2.05-13.7)]. Using the 95th percentile of controls as cut-off (P95=4120 per μL), the adjusted odds ratio was 2.20 (1.01-4.79) for individuals with MPs>P95 compared with those having MPs≤P95. After exclusion of individuals with antiphospholipid antibodies and hyperhomocysteinemia, the interaction between MPs>P95 and thrombophilia increased the VTE risk from 1.63 (0.60-4.50) to 6.09 (1.03-36.1).Conclusions: High levels of circulating MPs are a possible independent risk factor for VTE.

Plasma ADAMTS13, von Willebrand Factor (VWF) and VWF Propeptide Profiles in Patients with DIC and Related Diseases

2011-11-09

Abstract: ADAMTS13, endothelial von Willebrand factor (VWF) and related proteins are involved in the pathogenesis of some life threatening systemic thrombotic coagulopathies. Changes of plasma ADAMTS13 activity in thrombotic thrombocytopenic purpura (TTP) is well known but is also involved in septic disseminated intravascular coagulation (DIC). Here we investigated the ADAMTS13 activity, VWF and VWF propeptide (VWFpp) antigens in 69 patients with DIC, 143 with non-DIC, 21 with thrombotic thrombocytopenic purpura (TTP) and 23 with atypical hemolytic uremic syndrome (aHUS) for diagnosis of DIC.The plasma ADAMTS13 activity was significantly low in patients with DIC, and the plasma levels of VWF and VWFpp antigens, were the highest in these patients, but there were no significant differences in the plasma VWFpp levels between the patients with DIC and those with aHUS. The difference in the plasma ADAMTS13 activity, the VWF and VWFpp antigens between DIC and non-DIC cases was significant in those with infectious and malignant diseases, but the difference in the VWFpp/ VWF ratio were significant only in subjects with infectious diseases. As an indicator for prognosis, the plasma levels of VWFpp were significantly higher in non-survivors than in survivors. Then, VWFpp/ VWF ratio and VWFpp/ADAMATS13 ratio will be potent informative indicators in DIC.These findings suggest that ADAMTS13/VWF profiles may have important roles in the pathogenesis of DIC, and that ADAMTS13 and VWFpp are useful indicators for the diagnosis and prognosis of DIC.

The inhibition of the integrin VLA-4 in MV3 melanoma cell binding by non-anticoagulant heparin derivatives

2011-11-18

Abstract: Introduction: The integrin VLA-4-mediated binding is important for the metastatic dissemination of melanoma cells. Recently we found that heparin possesses a binding capacity to VLA-4. This could contribute to the heparin function to attenuate metastasis in a selectin-dependent manner. Aiming to a purposive, anti-adhesive heparin application, structural requirements of heparin for VLA-4 recognition have to be elucidated.Materials and methods: A series of non-anticoagulant heparin derivatives were investigated concerning their inhibitory capacities for VLA-4 mediated binding of human melanoma MV3 cells to VCAM-1 under physiological flow conditions in vitro. A surface acoustic wave biosensor was applied to detect kinetic constants of selected derivatives binding to both, VLA-4 or P- and L-selectin.Results: Experimental metastasis of MV3 cells in mice confirmed the relevance of VLA-4 for metastatic dissemination. LMWHs (enoxaparin, tinzaparin) efficiently blocked VLA-4 cell binding, dominantly via the integrin`s α-chain. Desulfation at 2-O-position, N-acetylation or a size smaller than tetradecasaccharide disfavoured VLA-4 inhibition. Glycol-splitting of heparin and thus higher chain flexibility is a tolerable parameter. A derivative with 50% 6-O-desulfation appeared promising and exceeded tinzaparin in VLA-4 inhibition, both compounds displayed binding affinities to VLA-4 in the low micromolar range.Conclusions: These findings provide structure-activity relationships for heparin VLA-4 binding, which partly differ from P- and L-selectin requirements. The data confirm that anti-coagulative and anti-adhesive function of heparin can be distinguished favouring applications of non-anticoagulant heparins in antimetastatic approaches without the risk of bleeding complications. The 50% 6-O-desulfated heparin-derivative appears promising to further evaluate the interference with selectin and VLA-4 binding functions in vivo.

Polymorphisms of PAI-1 and platelet GP Ia may associate with impairment of renal function and thrombocytopenia in Puumala hantavirus infection

2011-12-02

Abstract: Introduction: Puumala virus (PUUV) infection is a viral hemorrhagic fever with renal syndrome (HFRS) characterized by thrombocytopenia and acute impairment of renal function. We aimed to assess whether genetic polymorphisms of platelet antigens together with those of von Willebrand factor (VWF) and plasminogen activator inhibitor (PAI-1) correlate with disease severity.Patients and methods172 consecutive hospital-treated patients with serologically confirmed acute PUUV infection were included. Platelet glycoprotein (GP) IIIa T>C (rs5918), GP Ia T>C (rs1126643), GP Ib C>T (rs6065), GP VI T>C (rs1613662), VWF A>G (rs1063856) and PAI-1 A>G (rs2227631) were genotyped. The associations of the rarer alleles with variables reflecting the severity of the disease were analyzed.Results: PAI-1G-carriers had higher maximum creatinine level compared with the non-carriers (median 213μmol/l, range 60–1499μmol/l vs. median 122μmol/l, range 51–1156μmol/l, p=0.01). The GG-genotypes had higher creatinine levels than GA- and AA-genotypes (medians 249μmol/l, 204μmol/l and 122μmol/l, respectively, p=0.03). Polymorphisms of GP VI and VWF associated with lower creatinine levels during PUUV infection. The minor C-allele of GP Ia associated with lower platelet counts (median 44×109/l, range 20–90×109/l vs median 64×109/l, range 3–238×109/l; p=0.02).Conclusions: Polymorphism of PAI-1, a major regulator of fibrinolysis, has an adverse impact on the outcome of kidney function in PUUV-HFRS. Platelet collagen receptor GP Ia polymorphism associates with lower platelet count.

Influence of cytochrome 2C19 allelic variants on on-treatment platelet reactivity evaluated by five different platelet function tests

2011-12-12

Abstract: Background: The antiplatelet effect of clopidogrel has been linked to cytochrome P450 2C19 (CYP2C19) carrier status. The presence of loss of function and gain of function variants were found to have a gene-dose effect on clopidogrel metabolism. However, genotyping is only one aspect of predicting response to clopidogrel and several platelet function tests are available to measure platelet response.Patients and methodsWe studied the influence of CYP2C19 allelic variants on on-treatment platelet reactivity as assessed by light transmission aggregometry (LTA), the VerifyNow P2Y12 assay, the VASP assay, multiple electrode aggregometry (MEA), and the Impact-R in 288 patients after stenting for cardiovascular disease. Allelic variants of CYP2C19 were determined with the Infiniti® CYP450 2C19+ assay and categorized into four metabolizer states (ultrarapid, extensive, intermediate, poor).Results: Platelet reactivity increased linearly from ultrarapid to poor metabolizers using the VerifyNow P2Y12 assay (P =0.04), the VASP assay (P=0.02) and the Impact-R (P=0.04). The proportion of patients with high on-treatment residual platelet reactivity (HRPR) identified by LTA, the VerifyNow P2Y12 assay and the VASP assay increased when the metabolizer status decreased, while no such relationship could be identified for results of MEA and Impact-R. The presence of loss of function variants (*2/*2, *2-8*/wt, *2/*17) was an independent predictor of HRPR in LTA and the VASP assay while it did not reach statistical significance in the VerifyNow P2Y12 assay, MEA, and the Impact-R.Conclusion: Depending on the type of platelet function test differences in the association of on-treatment platelet reactivity with CYP2C19 carrier status are observed.

Pharmacodynamic assessment of a novel P2Y12 receptor antagonist in Japanese patients with coronary artery disease undergoing elective percutaneous coronary intervention

Hiroyoshi Yokoi, Takeshi Kimura, Takaaki Isshiki, Hisao Ogawa, Yasuo Ikeda — 2011-12-19

Abstract: Introduction: Numerous reports have shown that prasugrel shows a rapid and consistent antiplatelet effect among European and US patients. Previous studies suggest that prasugrel might be expected to achieve an adequate antiplatelet effect in healthy Asian subjects, even at lower doses than those assessed in the TRITON-TIMI 38 study. In this study, the antiplatelet effect of prasugrel was evaluated in Japanese coronary artery disease (CAD) patients undergoing percutaneous coronary intervention (PCI).Methods and results: Eighty-four patients were randomized into four treatment groups: prasugrel 10/2.5mg (loading dose [LD]/maintenance dose [MD]), 15/3.75mg or 20/5mg, and clopidogrel 300/75mg. The LD of each regimen was administered the day before PCI, followed by 28-day MD on aspirin background therapy (81–100mg). Antiplatelet effects were evaluated by light transmission aggregometry and VASP assay.The mean inhibition of platelet aggregation (IPA) induced by 20μM of adenosine diphosphate at 4hours after LD was higher among the prasugrel 10/2.5mg, 15/3.75mg and 20/5mg groups compared with the clopidogrel group (12.3%, 20.9%, 29.8% vs. 8.4%, respectively). The proportion of subjects with an IPA of <10% on Day 28 was lower among the prasugrel 15/3.75mg, and 20/5mg groups than in the clopidogrel group (0%, 6.3% vs. 15.8%, respectively). No “major” or “clinically relevant non-major” bleeding was observed.Conclusions: Prasugrel 15mg LD/3.75mg MD or higher doses was well tolerated and achieved a more rapid, higher and consistent antiplatelet effect than clopidogrel in Japanese CAD patients undergoing PCI.

Lack of association between polymorphisms in the interleukin-1 gene cluster and familial thrombophilia

Luke Marsden, Angela Cox, Mike Makris, Martina E. Daly — 2011-08-12

Abstract: Introduction: Inflammation and venous thrombosis are intimately linked, and there is evidence that levels of inflammatory cytokines influence risk of venous thrombosis. We investigated the hypothesis that allelic variation within the IL-1 gene cluster, which encompasses the genes encoding the inflammatory cytokines IL-1α and IL-1β and the competitive IL-1 receptor antagonist, is associated with venous thrombosis among patients with heritable thrombophilia.Subjects and Methods: Genomic DNA samples from 181 index cases with heritable thrombophilia and 323 control subjects were genotyped for four SNPs, and four microsatellite markers located within the IL-1 gene cluster. The distributions of SNP genotypes and of microsatellite marker alleles were then compared between the patient and control groups.Results: There was no significant difference in the distribution of alleles between the patients and control subjects for any of the four microsatellite loci studied. Likewise, the distribution of genotypes for each of the four SNPs investigated was similar among the cases and control subjects. Haplotype analysis showed no difference in the estimated frequencies of any of the IL-1 gene cluster haplotypes between the patients and control subjects.Conclusions: Our findings in this study suggest that inherited variation within the IL-1 gene cluster is not associated with thrombosis among patients with heritable thrombophilia and that alterations in inflammatory cytokines encoded by loci in the IL-1 gene cluster are more likely to occur as a result, rather than a cause, of venous thrombosis.

Determination of alpha-2-macroglobulin complexes by a new immuno-activity assay

Helen M. Atkinson, Nagina Parmar, Leslie R. Berry, Anthony K.C. Chan — 2011-08-17

Abstract: Introduction: Alpha-2-macroglobulin (α2M) is a broad specificity protease inhibitor which impacts several hemostatic pathways. Selective detection of various α2M complexes may be useful to define markers for the status of different hemostatic components. We present proof of principle for a novel assay to quantitatively measure α2M in complex with a variety of hemostatic factors.Materials and Methods: The assay makes use of the fact that α2M entraps proteases within a molecular “cage”, leaving them inaccessible to macromolecular substrates while retaining functionality against small synthetic substrates. Wells coated with anti-α2M antibodies were used to isolate the complexes from buffer or plasma, followed by detection of specific proteases with chromogenic substrates. Macromolecular inhibitors were added to eliminate signal from any unbound proteases.Results: Calibration curves constructed with purified protease-α2M complexes were sigmoidal in nature, as is typical with immuno-assays. The specificity of signal production was confirmed with inhibitors that target either free protease, or both free and α2M-bound protease. The detection range of the assay was dependent on the protease being measured, and the surrounding matrix. Interference in detection of complexes in plasma was found to be caused, in part, by free α2M. Thrombin-α2M complexes were quantified in adult and newborn plasma following induction of thrombin generation and found to be significantly higher in adults, likely due to higher prothrombin levels.Conclusions: This assay provides a versatile platform method for quantification of multiple protease-α2M complexes. It may prove useful for mechanistic in vitro studies of hemostatic pathways, and potentially for clinical applications.

Leptin upregulates tissue factor expression in human breast cancer MCF-7 cells

2011-08-16

Abstract: Introduction: Obesity is a risk factor for both cardiovascular disease and cancer development. Leptin, a cytokine produced by adipose tissue, controls different processes in peripheral tissues, including cancer development and thrombotic disorders in patients with a variety of clinical disorders. Tissue factor (TF), the trigger of blood clotting, is abundant in the adipose tissue.Since TF, often expressed by cancer cells, is considered a hallmark of cancer progression, we investigated whether leptin could modulate TF in the human metastatic breast carcinoma cell line MCF-7.Materials and Methods: MCF-7 cells were incubated with or without the different reagents at 37°C. At the end of incubation, cells were tested for procoagulant activity by a one-stage clotting assay, TF and TNF-α antigen levels and mRNA by ELISA and real-time RT-PCR, respectively. Leptin receptor was studied by FACS.Results: Both TF activity and antigen constitutively expressed by MCF-7 were significantly increased by leptin in a dose-dependent fashion. TF mRNA levels were also enhanced indicating that leptin exerts its effect at the transcription level. The effect of leptin was specific and required binding to its receptor (Ob-R), which was found on the surface of the cells, since antibodies against leptin and Ob-R completely prevented TF expression upregulation.In addition, leptin enhanced both TNF-α mRNA synthesis and secretion from MCF7. An anti-TNF-α MoAb completely abolished the leptin-induced TF expression.Conclusions: These data support the hypothesis that leptin, by its upregulation of TF, possibly mediated by TNF-α synthesis, may contribute to processes underlying both cancer and vascular cell disorders.

Pleural ELFA D-dimer assay: A surrogate marker for malignant pleural effusion

Alona Matveychuk, Gloria Rashid, Ziva Fridman, Alexander Guber, David Shitrit — 2011-08-29

Abstract: Background: Malignant pleural effusion is associated with enhanced fibrinolysis. However, no data are available concerning the precise role of pleural D-dimer assay in pleural effusion. We therefore assessed the role of pleural D-dimer assay in predicting malignant pleural effusion.Patients and Methods: A prospective laboratory investigation was conducted in a tertiary care teaching hospital. The study included consecutive patients with pleural effusion who presented at the Pulmonary Department between November 2009 and May 2010. Blood and pleural D-dimer levels were measured by Enzyme Linked Fluorescent assay (ELFA). The results were correlated with the clinical, laboratory, and radiological findings, and with the final diagnosis of the pleural fluid.Results: A total of 103 patients with pleural effusion were included in the study. The Pleural ELFA D-dimer results were found to be positively correlated with pleural etiology of malignancy (p=0.0001). Pleural etiology was also correlated with pleural LDH, pleural protein, pleural PH, pleural glucose, pleural and blood CRP, but not with ADA. In a binary logistic regression, only the pleural ELFA D-dimer assay was a significant predictor of the malignant pleural effusion (odds ratio 1.007; 95% confidence interval 1.002-1.012; p=0.007). The area under the receiver operating characteristics curve for malignancy was 0.79. A D-dimer level of 146mg/ml had a sensitivity of 82% and a specificity of 74%.Conclusions: We found high D-dimer levels among malignant pleural effusion. D-dimer might be useful as a simple, noninvasive, surrogate marker for malignant pleural effusion.

Pentadecapeptide BPC 157 reduces bleeding time and thrombocytopenia after amputation in rats treated with heparin, warfarin or aspirin

2011-08-16

Abstract: Recently, in rat abdominal aorta terminoterminal-anastomosis the stable gastric pentadecapeptide BPC 157 prevents obstructive thrombus formation and rapidly destroys already formed obstructive thrombus. Also, BPC 157 wound healing may signify the clot as conductive matrix or “scaffold” to speed up wound healing process, and decrease bleeding. Here, in rats, BPC 157 (10μg/kg, 10ng/kg) improved always reduced bleeding time and amount of bleeding after (tail) amputation only, heparin (250mg/kg, 25mg/kg, 10mg/kg i.v.), warfarin (1.5mg/kg i.g. once daily for 3 consecutive days), aspirin (0.1g/kg i.g. (once daily/3 consecutive days) or 1.0g/kgi.p. once), and amputation associated with those agents application. BPC 157 counteracting regimens (i.v., i.p., i.g. (immediately after any challenge)) correspondingly follow the route of bleeding-agents application. All heparin-, warfarin-, and aspirin-rats and normal-rats that received BPC 157 exhibited lesser fall in platelets count. BPC 157 attenuated over-increased APTT-, TT-values in 10mg/kg heparin-rats, but did not influence heparin activity (anti-Xa test). Indicatively, unless counteracted in BPC 157 rats, excessive bleeding-acute thrombocytopenia (<20% of initial values in heparin-rats) approaches substantial fall in platelets count known in type II HIT. Also, BPC 157 markedly prolongs the survival time (heparin-rats, 25mg/kg, right foot amputation).

The SISET incorrectly cited the JAAM DIC scoring system

Satoshi Gando — 2011-12-22

We read with interest the recent issue of Thrombosis Research from the Italian Society for Haemostasis and Thrombosis (SISET) about developing guidelines for clinical practice regarding the diagnosis and treatment of disseminated intravascular coagulation (DIC) . We greatly appreciate their evaluation of the scoring system of the Japanese Association for Acute Medicine (JAAM) . However, we deeply regret that the SISET incorrectly cited the JAAM DIC scoring system. The diagnostic scores for DIC reported for the JAAM in Table 1 are actually part of an old scoring system that included the fibrinogen level as a diagnostic item and where a total score ≥5 was needed to establish a DIC diagnosis .

The SISET incorrectly cited the JAAM DIC scoring system

2012-01-19

Dr Gando suggests to replace the JAAM score as currently reported in Table 1 of the Guidelines of the Italian Society for Haemostasis and Thrombosis (SISET) on the diagnosis and treatment of disseminated intravascular coagulation (DIC) with the revised version of the same score whose prognostic value has been evaluated in two publications .

Sensitivity of discharge diagnosis ICD-9 codes for pediatric venous thromboembolism is greater than specificity, but still suboptimal for surveillance and clinical research

2011-11-21

The poor reliability (specifically, low specificity) of International Classification of Diseases, Ninth Edition (ICD-9) codes in pediatric vascular disease has recently been established for arterial ischemic stroke (AIS) . Venous thromboembolism (VTE) is also a significant cause of morbidity and mortality in children. Beyond the neonatal period, its incidence in childhood (estimated at 4.9 per 100,000 children per year) is higher than that of AIS, and appears to have risen dramatically in the past several years .

Absence of JAK2V617F mutation in Chinese deep vein thrombosis patients without myeloproliferative neoplasms

Bingfei Dong, Yang Zhang, Xueqi Fu, Guangyi Wang — 2011-12-26

Deep vein thrombosis (DVT) is one of the most common health disorders and leads to significant morbidity and mortality. The reported annual incidence of venous thromboembolism (VTE) is approximately 1:1,000 in the United States . Acquired and genetic risk factors are involved in DVT development and progression. Acquired factors include increased age, obesity, pregnancy, surgery, trauma, malignancies, immobilization and oral contraceptives. The genetic factors involve mutations in Factor V Leiden, antithrombin, protein C, and protein S. In addition, United States Thrombosis and Hemostasis Centers pilot sites program has been established to generate the comprehensive catalog of genomic abnormalities that mediate thrombosis . However, potential genetic risk factors remain unknown.

Monitoring unfractionated heparin with APTT: A French collaborative study comparing sensitivity to heparin of 15 APTT reagents

2011-12-12

Although unfractionated heparin (UFH) has been replaced by low molecular weight heparin in many indications, it remains the anticoagulant of choice for selected patient groups. The most widely used test for UFH monitoring remains the activated partial thromboplastin time (APTT). Based on a retrospective study performed in the 1970s , a therapeutic APTT range of 1.5-2.5 times control has gained wide acceptance, but large differences in the responsiveness of several different test reagents to UFH were later recognized . Because of this inter-reagent variability, recommendations were later issued suggesting that therapeutic APTT ranges should be defined in each laboratory according to the reagent used . Thus, individual laboratories should define an APTT therapeutic range corresponding to a UFH concentration of 0.3-0.7IU/ml as assessed by anti-factor Xa measurement. With this calibration, APTT measurement is considered as a surrogate assay for UFH concentration. Before making choice of an APTT reagent, it is therefore important to have information on its sensitivity to UFH. Unfortunately, available data concern only a few APTT reagents and most modern reagents have not been evaluated . In order to gain insight into this topic, we compared sensitivity to UFH of 15 APTT reagents currently available in France in 2010. To the best of our knowledge, this is the largest comparison of currently used APTT reagents performed so far.

A case series of five episodes of massive LMWH non-fatal self-induced overdose in a single patient

Jacob Odeberg, Anders Carlsson, Caroline Hällsjö-Sander, Anna Ågren — 2012-01-19

Low molecular weight heparins (LMWH) are frequently used for the prophylaxis and treatment of venous thromboembolic diseases (VTE). Their subcutaneous administration in fixed doses has made possible the treatment of thromboembolic diseases in outpatient settings. Self-induced massive overdoses have recently been described in case reports, with various treatments and outcomes . Thus, the management of such episodes is not clarified. Protamine is recommended by the American College of Chest Physicians for the reversal of LMWH in about the same doses that is established for reversal of the effect of heparin (unfractioned heparin, UFH). However, in contrast to heparin, LMWH is only partly neutralized by protamine . Furthermore, in massive overdose, the amounts of protamine needed to neutralize LMWH would far exceed 50mg, which is the maximal single dose recommended . In a recent case report on an overdose of 212,500IU of the LMWH dalteparin a bolus dose of 500mg reversed the LMWH effect only partially and transiently (measured as normalized APTT and anti-Xa) . However, no bleeding complication was noted, in accordance with other reports . This may indicate that a conservative approach in the management of LMWH overdose might be appropriate in patients without symptoms. Such an approach is supported by our present report describing very large overdoses of dalteparin in a single patient. However, our report also demonstrates that the presence of trauma or a locus minoris for bleeding should warrant a more active management.

Evaluation of effectiveness of thrombolytic therapy of pulmonary embolism

Orhan Gokalp, Ismail Yurekli, Banu Lafci, Haydar Yasa, Ali Gurbuz — 2012-02-13

We congratulate the authors on their successful study . The results using the method, “catheter-directed ultrasound-accelerated thrombolysis (USAT),” were reported, albeit relatively new, to be satisfactorily implemented in the treatment of acute pulmonary embolism . Evaluation of the effectiveness of the treatment in studies has generally been based on the clinical improvement along with the degree of thrombolysis confirmed on CT in the clot in the pulmonary artery . In the present study, however, Engelhardt et al. assessed the right-left ventricular diameter ratios (RV/LV) on CT, detecting a decrease in the ratio depending on the right ventricular shrinkage following the procedure . Based on our experience with the USAT method in two patients in our clinic, we also believe that symptoms are likely to exhibit a complete resolution and that the findings associated with the right ventricular failure especially with RV/LV ratio may improve even if the CT scan fails to reveal complete thrombolysis in the pulmonary arterial clot. As for our two cases, complete thrombolysis confirmed on CT was achieved in 1, while only partial lysis was documented in the other. However, we should also note complete resolution of the symptoms and decrease in RV/LV ratios in the two patients. In conclusion, we believe that the assessment of RV/LV ratio should dictate the effectiveness of the USAT therapy rather than the degree of thrombolysis in the clot impacted in the pulmonary artery.

Reply to “Evaluation of effectiveness of thrombolytic therapy of pulmonary embolism”

Tod C. Engelhardt — 2012-03-29

We thank you for your interest in our study. We agree that the RV/LV ratio should be considered an important measure of effectiveness for ultrasound-accelerated thrombolysis (USAT), or for other modes of acute PE therapy. Indeed, a number of studies report RV dilation (with elevated values of the RV/LV ratio) as a predictor of poor prognosis (including death), whereas regression of the dilated RV achieved by therapy allows improvement of such unfavorable outcomes . Furthermore, the recent 2011 AHA Scientific Statement proposes the RV/LV ratio of greater than 0.9 to be a key determinant of risk stratification of acute PE patients, according to which different levels of therapy are recommended . Supported by these findings, our study demonstrated the effectiveness of USAT by observing a reduction of the RV/LV ratio, as well as a complete resolution of symptoms, which are also evident in your patients.

Prevention of venous thromboembolism in patients with cancer: Guidelines of the Italian Society for Haemostasis and Thrombosis (SISET)1

2011-10-03

Abstract: Background: Prevention of venous thromboembolism (VTE) in cancer patients remains controversial in most clinical settings.Purpose: The Italian Society for Haemostasis and Thrombosis (SISET) commissioned a project to develop clinical practice guidelines for the prevention of VTE in patients with malignancy.Methods: Key questions concerning the prevention of VTE in patients with malignancy were formulated by a multidisciplinary working group consisting of experts in clinical medicine and research. After a systematic review and discussion of the literature, recommendations were formulated and graded according to the supporting evidence. For those questions for which the literature search did not find any definitive answers (due to absence of evidence, low quality evidence and/or contradictory evidence), a formal consensus method was used instead to issue clinical recommendations.Results: The search for “VTE prevention” resulted in 1021 citations; 69 articles were selected and 24 were used for drafting clinical recommendations. Four areas were graded A to C: 1) Need of prevention (pharmacological and/or mechanical) in cancer patients undergoing major abdominal or pelvic surgery and in 2) those with an acute medical disease requiring hospitalization and who are bedridden. Avoid prevention in 3) cancer patients with a central venous catheter and 4) those on chemotherapy, radiotherapy or hormonal therapy, except patients with multiple myeloma treated with thalidomide/lenalidomide plus high-dose dexamethasone, and those with gastrointestinal or lung cancer. Six areas were considered to be clinically important, but lacked evidence from the literature and thus required a formal consensus (grade D): 1) need of prevention during chemo- radiotherapy or hormonal therapy in patients with previous VTE; 2) optimal duration of pharmacological prevention in patients who are hospitalized/bedridden for acute medical illness; 3) optimal duration of pharmacological prevention in patients undergoing major surgery other than abdominal and pelvic; 4) optimal duration of pharmacological prevention in myeloma patients receiving thalidomide plus dexamethasone; 5) presence of cerebral metastasis as a contraindication to pharmacological prevention; 6) prevention in cancer patients undergoing surgery by laparoscopic procedures lasting>30min.Conclusion: Results of the systematic literature review and an explicit approach to consensus techniques have led to recommendations for the most clinically important issues in the prevention of VTE in cancer patients.

Diagnosis and treatment of disseminated intravascular coagulation: Guidelines of the Italian Society for Haemostasis and Thrombosis (SISET)

2011-09-19

Abstract: Background: The diagnosis and treatment of disseminated intravascular coagulation (DIC) remain extremely controversial.Purpose: The Italian Society for Thrombosis and Haemostasis commissioned a project to develop clinical practice guidelines for the diagnosis and treatment of DIC.Methods: Key questions about the diagnosis and treatment of DIC were formulated by a multidisciplinary working group consisting of experts in clinical medicine and research. After a systematic review and discussion of the literature, recommendations were formulated and graded according to the supporting evidence. In the absence of evidence, evidence of low quality, or contradictory evidence, a formal consensus method was used to issue clinical recommendations.Results and Conclusions: In suspected DIC, we suggest the use of the diagnostic scores ISTH (grade C), JMHW (grade C) or JAAM (grade D) over stand alone tests. The cornerstone of the management of DIC remains the treatment of the underlying triggering disease. We do not suggest the use of antithrombin (grade D), dermatan sulphate (grade D), gabexate (grade D), recombinant factor VIIa (grade D), activated protein C (grade D), thrombomodulin (grade B). The use of unfractionated heparin or low-molecular-weight heparin is not suggested except for thromboembombolic prophylaxis in patients a high risk who do not have active bleeding (grade D). In patients with severe sepsis/septic shock and DIC we suggest the use of human recombinant activated protein C (grade D). In patients with DIC and active bleeding we suggest the use of transfusion therapy (platelets, plasma, cryoprecipitate) (grade D).

Unexplained infertility: Association with inherited thrombophilia

2012-03-16

Abstract: Introduction: Unexplained infertility represents one of the most common diagnoses in fertility care. Attention is being paid to the association between inherited thrombophilia and infertility causes. In this study we investigated the prevalence of inherited thrombophilia according to infertility causes.Materials and methods: We studied Prothrombin gene G20210A mutation, Factor V Leiden, deficiencies in protein S and C and antithrombin in 930 Caucasian infertile women referred to Fertility Center of the Department of Sciences for Woman and Child's Health, University of Florence, of whom 230 with unexplained, 195 female and 283 male infertility, and in 240 women who have conceived naturally without hormonal stimulation therapy.Results: A significant relationship between inherited thrombophilia [OR 95%CI 1.97 (1.05-3.68), p=0.03] and unexplained infertility was observed, whereas no association between thrombophilia and female and male infertility was found. Significantly higher prevalence of prothrombin gene mutation in unexplained infertile women in comparison to that observed in fertile women was observed (5.7% vs 2.1% p=0.04); the prevalence of the other thrombophilia determinants was higher, even if not significantly, in the unexplained infertile group.Conclusions: This study demonstrates the relationship between inherited thrombophilia and unexplained infertility, thus suggesting the contribution of genetic components in modulating unexplained infertility, behind anovulation, male and tubal factor.

Performance of the Pulmonary Embolism Rule-out Criteria (the PERC rule) combined with low clinical probability in high prevalence population

2012-03-19

Abstract: Introduction: PERC rule was created to rule out pulmonary embolism (PE) without further exams, with residual PE risk<2%. Its safety is currently not confirmed in high PE prevalence populations even when combined with low clinical probability assessed by revised Geneva score (RGS). As PERC rule and RGS are 2 similar explicit rules with many redundant criteria, we hypothesized that the combination of PERC rule with gestalt clinical probability could resolve this limitation.Methods: We collected prospectively documented clinical gestalt assessments and retrospectively calculated PERC rules and RGS from a prospective study of PE suspected patients. We analyzed performance of combinations of negative PERC with low clinical probability assessed by both methods in high overall PE prevalence population.Results: Among the final study population (n=959), the overall PE prevalence was 29.8%. Seventy-four patients (7.7%) were classified as PERC negative and among them, 4 patients (5.4%) had final diagnosis of PE. When negative PERC was combined with low pretest probability assessed by RGS or gestalt assessment, PE prevalence was respectively 6.2% and 0%. This last combination reaches threshold target of 2% and unnecessary exams could easily have been avoided in this subgroup (6%). However, it confidence interval was still wide (0%; CI 0–5).Conclusions: PERC rule combined with low gestalt probability seems to identify a group of patients for whom PE could easily be ruled out without additional test.A larger study is needed to confirm this result and to ensure safety.

Derivation and validation of a multivariate model to predict mortality from pulmonary embolism with cancer: The POMPE-C tool

2012-04-04

Abstract: Background: Clinical guidelines recommend risk stratification of patients with acute pulmonary embolism (PE). Active cancer increases risk of PE and worsens prognosis, but also causes incidental PE that may be discovered during cancer staging. No quantitative decision instrument has been derived specifically for patients with active cancer and PE.Methods: Classification and regression technique was used to reduce 25 variables prospectively collected from 408 patients with AC and PE. Selected variables were transformed into a logistic regression model, termed POMPE-C, and compared with the pulmonary embolism severity index (PESI) score to predict the outcome variable of death within 30days. Validation was performed in an independent sample of 182 patients with active cancer and PE.Results: POMPE-C included eight predictors: body mass, heart rate >100, respiratory rate, SaO2%, respiratory distress, altered mental status, do not resuscitate status, and unilateral limb swelling. In the derivation set, the area under the ROC curve for POMPE-C was 0.84 (95% CI: 0.82-0.87), significantly greater than PESI (0.68, 0.60-0.76). In the validation sample, POMPE-C had an AUC of 0.86 (0.78-0.93). No patient with POMPE-C estimate ≤5% died within 30days (0/50, 0-7%), whereas 10/13 (77%, 46-95%) with POMPE-C estimate >50% died within 30days.Conclusion: In patients with active cancer and PE, POMPE-C demonstrated good prognostic accuracy for 30day mortality and better performance than PESI. If validated in a large sample, POMPE-C may provide a quantitative basis to decide treatment options for PE discovered during cancer staging and with advanced cancer.

A novel ENG mutation causing impaired co-translational processing of endoglin associated with hereditary hemorrhagic telangiectasia

2012-03-05

Abstract: Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant vascular dysplasia caused by mutations in mainly the endoglin gene (ENG) or activin-like kinase receptor 1 (ALK1) gene (ACVRL1). We investigated the molecular basis of HHT in a Japanese patient, and identified a novel missense mutation in ENG (c.38T>A, p.Leu13Gln) located in the signal peptide's hydrophobic core, but not in ACVRL1. In experiments in COS-1 cells, the Leu13Gln (L13Q) mutant endoglin appeared to be expressed as a precursor form, probably due to impaired protein processing. Flow cytometry analyses of the COS-1 cells transiently expressing recombinant endoglins revealed that the wild-type endoglin was detected on the cell surface, but the L13Q mutant was not. We also analyzed expression patterns of the recombinant endoglins by immunofluorescent staining, and found that the wild-type co-localized with the endoplasmic reticulum (ER), but the L13Q mutant did not. These results implied that the L13Q mutant endoglin fails to insert into the ER, probably due to destruction of the hydrophobic core structure in the signal peptide to be recognized by signal recognition particles. Thus, the Leu13 in the signal peptide of endoglin might be essential for correct protein processing through the ER and cell-surface expression. Taken together, the novel c.38T>A mutation in ENG would impair co-translational processing of the endoglin, and could be responsible for HHT in this patient.

Pennogenin tetraglycoside stimulates secretion-dependent activation of rat platelets: Evidence for critical roles of adenosine diphosphate receptor signal pathways

2012-03-09

Abstract: Background: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been demonstrated to promote hemostasis in vivo and induce platelet aggregation in vitro. Pennogenin tetraglycoside (Tg) has been identified as one of the active ingredients in TSSPs and can induce rat platelet aggregation.Objective: To investigate the functional role of Tg in platelets and the signaling pathway mechanisms which mediate Tg-induced platelet aggregation.Methods and results: Using scanning electron microscopy, the turbidimetric method and flow cytometry, we demonstrated that Tg induces shape change and concentration-dependently induces aggregation, dense granule secretion and a-granule secretion in rat platelets. The activation characteristics were comprehensively confirmed using transmission electron microscopy. Apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, completely inhibited Tg-induced platelet aggregation, which was not sensitive to indomethacin or SQ29548 inhibition. Furthermore, ADP receptor antagonists inhibited Tg-induced a-granule secretion, and blockade of the P2Y1 receptor prevented Tg-induced platelet shape changes. Tg-induced dense granule secretion was not affected by ADP receptor antagonists or various various pharmacological inhibitors of the intracellular effectors involved in dense granule secretion signaling pathways.Conclusion: We identified that Tg directly induces platelet activation and demonstrated that Tg-induced platelet activation depends on dense granule secretion of ADP, which in turn activates the P2Y1 and P2Y12 receptor signaling pathways.

A novel anti-platelet peptide (Z4A5) potential for glycoprotein IIb/IIIa inhibits platelet aggregation

2012-03-26

Abstract: Introduction: Z4A5 is a novel peptide that inhibits platelet aggregation and formation of platelet thrombi, but the mechanism of its anti-platelet effects remains unknown. This study explores the anti-platelet effect and mechanism of Z4A5.Methods: We investigated the anti-platelet activity of Z4A5 on platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin (TH) in human platelet-rich plasma (PRP). Fibrinogen and PAC-1 binding to glycoproteinIIb/IIIa (GPIIb/IIIa) were measured by flow cytometry. In addition, we investigated the integrin specificity of Z4A5 in attachment and detachment assays using human umbilical vein endothelial cells (HUVEC) and assessed the relative cell number using the MTT assay.Results: In vitro, Z4A5 inhibited ADP-, AA- and TH-induced human platelet aggregation with IC50 values of 0.46±0.05μM (n=10), 0.23±0.05μM (n=10) and 0.21±0.02μM (n=10), respectively. Z4A5 inhibited fibrinogen, and PAC-1 bound to platelet GPIIb/IIIa with IC50 values of 0.48±0.07μM (n=8) and 0.63±0.12μM (n=6), respectively. Z4A5 failed to inhibit αVβ3 integrin-mediated HUVEC attachment to vitronectin and did not cause any significant detachment of HUVEC monolayer when compared with the controls.Conclusions: Z4A5 is a potential anti-platelet drug that inhibits fibrinogen binding to GPIIb/IIIa, but does not affect the structurally similar integrin αVβ3.

Unfractionated heparin promotes LPS-induced endothelial barrier dysfunction: A preliminary study on the roles of angiopoietin/Tie2 axis

Xu Li, Zhen Zheng, Yiran Mao, Xiaochun Ma — 2012-03-28

Abstract: Introduction: Heparins, including unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), are anticoagulants approved as a treatment for severe sepsis, which can also prevent apoptosis and inflammation. The aim of this study was to investigate whether UFH prevents vascular leakage induced by lipopolysaccharide (LPS) and to define the role of angiopoietin (Ang)/Tie2 signaling pathway since LPS is usually used to mimic the initiation of sepsis.Methods: Human pulmonary microvascular endothelial cells (HPMECs) were pretreated with UFH (0.1U/ml-10U/ml), 15min prior to stimulation with LPS (10μg/ml). Those samples not receiving LPS or UFH received an equal volume of Phosphate-buffered saline (PBS). Cells were cultured under various experimental conditions for 2h, 6h or 12h for analysis.Results: 1) Pretreatment with UFH significantly reduced HPMEC permeability compared with LPS-stimulated groups; 2) Pretreatment with UFH decreased the formation of stress fiber and intracellular gaps induced by LPS; 3) UFH significantly up-regulated gene expression of Tie2 and Ang-1 but down-regulated Ang-2 in HPMECs; 4) UFH prevented LPS-induced decrease in the level of ZO-1.Conclusion: This study demonstrates that UFH enhances endothelial barrier function and Ang/Tie2 axis probably represents one of the mechanisms by which UFH exerts its protective effect.

Reduced clot-stability during the first 6hours after aneurysmal subarachnoid haemorrhage – a prospective case-control study

Carl C. Larsen, Benny Sørensen, Jørn D. Nielsen, Jens Astrup — 2012-03-05

Abstract: Introduction: Early rebleeding is an important cause of death and disability following aneurysmal subarachnoid haemorrhage (SAH). Recent studies have shown that 50-90% of the rebleedings occurred within the first 6hours after the primary bleeding. The mechanism leading to rebleeding remains to be established. In the present prospective case-control study we hypothesize that patients with SAH develop a coagulopathy characterized by reduced clot stability during the early period after the initial bleeding.Methods: Patients with aneurysmal SAH was studied with a dynamic clot lysis assay and markers of fibrinolysis and clot stabilizers in blood samples taken within and after 6hours after onset of bleeding. Results were compared with blood samples from age and gender matched healthy controls.Results: 36 patients were enrolled, 26 patients had blood samples collected within 6hours after the initial bleeding whereas 10 patients had blood samples taken later than 6hours after the initial bleeding. Patients demonstrated significantly reduced clot stability during the first 6hours after initial bleeding. Fibrinolytic activity was increased during the first 6hours along with the inhibitors of fibrinolysis whereas the modulators of fibrinolysis were reduced or inactivated.Conclusion: During the first 6hours after SAH patients exhibit reduced clot-stability. Probably a consequence of activated fibrinolysis in combination with reduced or inactivated factor XIII and thrombin-activable fibrinolysis inhibitor.

Economic analysis of recombinant activated factor VII versus plasma-derived activated prothrombin complex concentrate in mild to moderate bleeds: Haemophilia registry data from the Czech Republic

2012-03-05

Abstract: Introduction: Several studies suggest that recombinant activated factor VII (rFVIIa) is more cost-effective than plasma-derived activated prothrombin complex concentrate (pd-aPCC) in haemophilia with inhibitors. However, most do not consider differences between treated patients. This study compared the pharmacoeconomics of rFVIIa versus pd-aPCC treatment of mild to moderate bleeds in inhibitor patients, taking co-variables into account.Methods: The HemoRec and HemIS registries capture exhaustive bleeding data in inhibitor patients in the Czech Republic. For each bleed, patient and bleed characteristics, treatment outcomes and bypassing agent use were retrospectively analysed, and direct costs of care per bleed calculated. Generalised Linear Model regression methods with cluster effect were employed to account for the possibility of several bleedings from the same patient.Results: There were 108 and 53 mild to moderate bleeds in the rFVIIa and pd-aPCC groups, respectively. Although re-bleeding rates were similar in both groups, deeper analyses revealed significant differences in time to bleed resolution: 93.8% of bleeds treated with rFVIIa were resolved within ≤12h, versus 60.4% with pd-aPCC (P<0.001). Mean total cost/bleed was lower with rFVIIa (336,852 [median, 290,696] CZK; €12,760 [11,011]) than pd-aPCC (522,768 [341,310] CZK; €19,802 [12,928]) (P=0.002). Results were maintained after controlling for potential co-variables (bleed nature, time to treatment, target joints).Conclusions: The lower total treatment costs per bleed with rFVIIa than pd-aPCC suggest that first-line rFVIIa is more cost-effective than pd-aPCC in mild to moderate bleeds. Time to bleed resolution was also significantly shorter with rFVIIa. These results were maintained when controlled for potential confounders.

Compartment-specific expression of plasminogen activator inhibitor-1 correlates with severity/outcome of murine polymicrobial sepsis

2012-03-01

Abstract: Introduction: Plasminogen activator inhibitor type 1 (PAI-1) co-induces septic coagulopathy. We aimed to characterize spatiotemporal PAI-1 gene/protein changes occurring in acute sepsis and tested whether PAI-1 fluctuations correlate with sepsis severity and early outcome.Materials and Methods: Female mice underwent cecal ligation and puncture (CLP) in three experiments. I: mild (23G needle) CLP to compare circulating PAI-1 to its organ gene expression within 0-24h. II: mild or severe (17G) CLP to asses differences in PAI-1 organ-specific expression and in coagulation/fibrinolysis. III: moderate (18G) CLP to characterize circulating PAI-1 in survivors (SUR), and to retrospectively compare it to dying (DIE) mice.Results: In mild sepsis, the trajectory of circulating PAI-1 (1089ng/ml peak at 24h) was identical to PAI-1 gene expression in the left cranial vena cava (LCVC; 39-fold peak at 24h). PAI-1 expression rise was immediate (60-fold at 6h) and sustained in the liver, but marginal in the kidney, lungs and heart. Body temperature decrease correlated with the PAI-1 expression increase in the liver (rho=−0.79), and blood (protein, rho=−0.53). Regardless of severity, PAI-1 gene expression remained unaltered except the LCVC where it was >3-fold higher in 17G (vs. 23G). Severe sepsis extended activated partial thromboplastin/pro-thrombin time and increased circulating PAI-1, while antithrombin and fibrinogen decreased at 6 and/or 24h (vs. 23G). Within 24h of death, circulating PAI-1 in DIE was >3-fold higher versus SUR.Conclusions: Polymicrobial sepsis caused a gradual circulating PAI-1 release and highly variable gene expression response pattern in organs. Only circulating PAI-1 and PAI-1 expression in the LCVC correlated with response severity and/or outcome.

Formation of the venous thrombus after venous occlusion in the experimental mouse model of metabolic syndrome

2012-03-29

Abstract: Introduction: The metabolic syndrome is considered to be a risk factor for the venous thromboembolism (VTE) as well as arterial thrombosis. Although obesity, hyperglycemia and dyslipidemia are considered to be important triggering factors, it is difficult to evaluate the relationship between VTE and the metabolic syndrome in a clinical study. Furthermore the mechanism of venous thrombosis initiation still remains elusive.Materials and Methods: 20min clamp of superior mesenteric vein was applied to 7w, 16w-old KK-Ay mouse and 16w-old B6J mouse (n=6 in each group), after de-clamp, the view of the mesenteric vein and intestinal submucosal venule were observed by the intravital microscopy.Results: Massive thrombi formed in the mesenteric vein in 16w-old KK-Ay mice, moderate thrombi formation was observed in 7w-old KK-Ay mice, while very few thrombi were observed in B6J mice. The first event in submucosal venule after de-clamp was the adhesion of leukocytes to the endothelium. Subsequently, leukocytes assembled and platelets covered the leukocyte cluster. These leukocyte-platelet aggregates move from the venule to the vein and finally formed a venous thrombus.Conclusion: Metabolic syndrome is a risk factor for venous thrombosis. Intravital microscopic examination revealed leukocyte and platelet recruitment to the venule in the early stages of venous thrombosis formation.

Monomeric C-reactive protein alters fibrin clot properties on endothelial cells

Rong Li, Meiping Ren, Mao Luo, Ni Chen, Zhuo Zhang, Bo Luo, Jianbo Wu — 2012-04-04

Abstract: Elevated plasma levels of C-reactive protein (CRP) are independently associated with increased risk of atherothrombosis. Several lines of evidence suggest that CRP has prothrombogenic effects on injured vessel wall(s) by enhancing tissue factor (TF) expression. Abnormal fibrin formation is correlated with increased thrombotic risk. However, the impact of localized, cell surface-driven in situ tissue factor generation by CRP on clot dynamics and fibrin architecture has not previously been evaluated. We examined the impact of native CRP and modified or monomeric CRP (mCRP) on the fibrin formation and structure in Human Umbilical Vein Endothelial Cells (HUVECs). Fibrin formation and structure were examined using laser scanning confocal microscopy. Incubation with mCRP on the cell surface had faster fibrin polymerization by the analysis of turbidimetry. Confocal microscopy of fibrin clots showed a significantly increased density in the treatment of mCRP compared with native CRP and control in the proximal versus distal relationship to the cell surface. The increased expression and activity of TF on the cell surface was observed by addition of mCRP. Blockage of tissue factor and lipid rafts significantly reduced the density of fibrin network produced by mCRP-stimulated endothelial cells. mCRP changes clot dynamics and alters fibrin architecture by enhancing TF on the endothelial cell surface. These results support the concept that elevated CRP levels may induce fibrinolytic resistance and endothelial dysfunction by altering fibrin clot structure.

Progestin-only contraception and venous thromboembolism

M.A. Blanco-Molina, M. Lozano, A. Cano, I. Cristobal, L.P. Pallardo, I. Lete — 2012-03-19

Abstract: Combined oral contraceptives (COC) are the most popular contraceptive method in developed countries. Since their introduction there have been numerous changes and modifications in its composition with the aim to improve safety and tolerability while maintaining contraceptive efficacy. Most of the changes have been conducted on the progestin component, since most of the combinations include ethinyl estradiol as oestrogen. One of the adverse effects of COC is the increased risk of venous thromboembolism (VTE) in two clinical forms of presentation: deep vein thrombosis or pulmonary embolism. This review details the changes in haemostasis induced by progestin-only contraceptives and the risk of VTE in women who utilize this type of contraception; the relationship with other risk factors such as thrombophilia; the interactions of these contraceptives with anticoagulant treatment and finally the eligibility criteria for the use of hormonal contraception in women with previous VTE or thrombophilia carriers.

Theragnostic performance of ex vivo testing of haemostatic intervention and clinical effect of recombinant factor VIIa and fibrinogen concentrate

Mariann Tang, Per Wierup, Kirsten Christiansen, Jacob Greisen, Benny Sørensen — 2012-04-04

During recent years, point-of-care (POC) thromboelastometry has been implemented to facilitate theragnostics as defined by prompt individualised diagnosis of coagulopathy and goal directed haemostatic therapy. Several studies report on successful outcomes and reduced use of allogeneic blood products following the introduction of thromboelastometry guided theranostic algorithms . Obvious advantages include near patient testing, speedy results and use of whole blood. Standard ROTEM assays demonstrate good performance in differentiating dysfunctional fibrin polymerisation, thrombocytopenia, hyperfibrinolysis, and heparin effect . However, occasionally the currently available theragnostic approaches fail to detect abnormalities and guide intervention.

Clinical and biological factors that contribute to thrombin generation in prothrombin G20210A carriers: A case–control study in a single Thrombophilia Center

Ana Marco, Concepción Brocal, Fernanda Martirena, Javier Lucas, Pascual Marco — 2012-04-04

Prothrombin gene mutation known as PTG20210A, first described in 1996 leads to an increased amount of prothrombin plasma levels . Approximately 1-2% of the general population is heterozygous for this prothrombin gene mutation and there is an estimated incidence of deep venous thrombosis of 1/1000 per year. The exact mechanism by which this mutation results in a thrombophilic state is still unclear . Marchiori et al. have reported a risk ratio (RR) for venous thrombosis disease (VTD) recurrence of 1.74 by the Mantel-Haenszel fixed effect method and a RR of 1.62 by the Der Simonion and Laird random effects model, comparing PTG20210A carriers vs non- carriers .

The meanings of DIC diagnostic criteria

Toshiaki Iba, The Japanese Association for Acute Medicine (JAAM) DIC study group — 2012-02-06

We read with interest the recent issue of Thrombosis Research from Singh RK et al. about the validation of the scoring systems for disseminated intravascular coagulation (DIC). We greatly appreciate their evaluation of the scoring system of the Japanese Association for Acute Medicine (JAAM) . However, there are some concerns to their study. Firstly, as written in the discussion, the sample size of this study is too small. While Gando's report included 273 cases from 13 emergency centers prospectively, Singh et al. included only 42 cases from the single institute. In addition, the usefulness for the discrimination of survival was demonstrated in Gando's report, while there was no such distinction in Singh's study. According to another prospective study, similar results as Gando's report were also demonstrated (). As mentioned in Singh's study by itself, one possible reason for this discrepancy is due to the low statistical power based on its small sample size. Therefore, we would like to introduce another recent larger prospective study, which included 413 cases. This study also demonstrated that the DIC diagnosis by JAAM criteria was related to poor outcomes .

Author's comments on Letter to editor "The meanings of DIC diagnostic criteria"

R.K. Singh, A.K. Baronia — 2012-03-05

We are thankful to Toshiaki Iba for his insightful and critical comments on our study . It's true as mentioned by Toshiaki Iba that too small sample size, high mortality and multidrug resistant pathogens might be responsible as to why disseminated intravascular coagulation (DIC) diagnosed by new Japanese Association for Acute medicine (JAAM) criteria was not found to be related to the poor outcome in our study. We also agree to his comments that perhaps our patient population was not representative of the cohort that is encountered in most of the ICUs where DIC scores had been previously studied. However, this cannot be the reason to discredit our findings for the simple reason that subsequent studies may also replicate similar results in “high mortality” prone patients infected by multidrug resistant organisms. In fact it might be a worthwhile observation suggesting that JAAM DIC criteria may fail to act as a predictor of mortality in a subset of critically sick patients. His interpretation regarding inclusion of the coagulation score in SOFA score is incorrect as it has been clearly mentioned in our study that it is not the case.