Bleeding Tendency In Patients Research Articles

Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements

Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one-stage clotting and two-stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty-six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time-based one-stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one-stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population.

Acquired factor V deficiency in a patient without evidence of a classical inhibitor

Acquired factor V deficiency in a patient without evidence of a classical inhibitor

Impairment of platelet retention rate in patients with severe aortic valve stenosis

BackgroundRecent reports revealed the presence of acquired von Willebrand syndrome type 2A in patients with aortic valve stenosis (AS). von Willebrand factor (vWF) has been shown to play a vital role in platelet adhesion. Therefore, we measured the platelet retention rates, which reflect platelet adhesion, in patients with severe AS. MethodsIn addition to echocardiography, routine blood screening tests were performed and the platelet retention rates were measured using collagen-coated bead columns in 21 patients with severe AS and in 21 control subjects. ResultsPatients with severe AS showed the maximum aortic valve pressure gradients of 110.9±22.7mmHg, and effective orifice areas of 0.59±0.20cm2. The results of routine blood tests in patients with severe AS were comparable to those of control subjects; however, the platelet retention rates in the AS patients (7.3±5.0%) were significantly lower than those in control subjects (30.5±11.8%, p<0.001). A significant negative correlation was observed between maximum aortic valve pressure gradients and platelet retention rates (r=−0.81, p<0.001). In 8 patients with severe AS, the platelet retention rates increased from 5.8±3.6% to 16.0±2.4% after aortic valve replacement (p<0.001). ConclusionThese findings suggest that impairment of platelet retention rate is seen in almost all patients with severe AS. Clinicians should be aware of the possibilities of vWF-mediated platelet dysfunction and bleeding tendency in patients with severe AS.

Severity of the Loss of High Molecular Weight Multimers Is Associated with Heyde Syndrome in Patients with Severe Aortic Stenosis.

AbstractAbstract 2230 Background:Heyde syndrome describes patients with severe aortic stenosis (AS) who have bleeding diathesis, especially gastrointestinal bleeding. Although acquired won Willebrand syndrome (AVWS) due to loss of the highest molecular weight multimers (HMWM) presents in most patients with severe AS, not all such patients develop Heyde syndrome. The goal of this study was to explore whether the severity of HMWM loss was significantly associated with Heyde syndrome. Methods:We first performed chart reviews on patients referred for double balloon enteroscopy (DBE) for GI bleeding or iron-deficiency anemia (GIB/FeDefic) in two time periods, from 2005–2008, and from 2009–2011. Next, patients who could have AVWS due to AS or other cardiovascular or hematologic disorders were identified. Laboratory tests including VWF antigen (VWF:Ag), VWF ristocetin cofactor activity or VWF latex immunoturgidic activity (VWF:Ltx) and/or VWF multimer analysis were performed as requested by physicians. From this population, patients with Heyde syndrome were identified. Their clinical and VWF laboratory testing results were compared to a separated group of patients with severe AS patients who had no clinically significant bleeding, but had matched gender and hemodynamic severity by echocardiogram (Matched AS Controls). These patients all had VWF:Ag, VWF:Ltx, and VWF multimers performed. In both Heyde syndrome patients and Matched AS controls, VWF multimer densitometry ratio of the HMWM (>15 mers) to low to intermediate multimers (2–15 mers) was assessed (VWF:multi ratio). Results:Overall, 437 patients underwent DBE for GIB/FeDef. Between 2005 and 2008, 1% of 318 patients had VWF testing. From 2009 to 2011, 23% of 119 patients were tested. Heyde syndrome was noted in 15 patients. Of the 437 patients, 134 patients (31%) likely had AVWS based on diagnoses of moderate-severe AS (30%), moderate to severe mitral regurgitation (25%), moderate to severe pulmonary hypertension (16%), prosthetic valve regurgitation or stenosis (13%), hypertrophic cardiomyopathy (10%), and hematologic disorders (4%). Among these 134 patients, 29 patients had VWF tests performed, and 22 had VWF multimer analysis. VWF antigen levels were similar in Heyde syndrome and Matched AS Controls (176±89% versus 155±77% respectively). Despite similar hemodynamic severity, more severe HMWM loss as indicated by lower VWF:Ltx/Ag and VWF:multi ratios was present in Heyde syndrome patients than matched AS controls (table, P<0.0007).Age, yearsMean gradient, mm HgVWF: Ltx/VWF:AgVWF:multi ratiosPFA-CADPHeyde (n=15)76±9†47±12†0.78±0.06*0.106±0.033*203±57†Matched AS Controls (n=25)78±1147±150.89±0.050.143±0.015174±121N=10 for VWF activity/antigen and PFA-CAPD in Heyde syndrome patients.*p<0.0007.†p=NS Conclusion:If AVWS is suspected in patients with GIB/FeDef, laboratory testing for VWF indices is important to confirm the diagnosis of AVWS. Despite similar hemodynamic severity, Heyde syndrome patients have more severe VWF HMWM loss indicated by significantly decreased VWF:Ltx/VWF:Ag and VWF:multi ratios. Conversely, though highly sensitive for AVWS, PFA-CADP does not predict bleeding tendency in patients with severe AS. Disclosures:No relevant conflicts of interest to declare.

Microparticle-Associated Thrombogenic Mechanism Might Compensate Bleeding Tendency in Patients with Myelodysplastic Syndromes.

Abstract 2821 Introduction:Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders with clonal bone marrow anomalies characterized by ineffective hematopoiesis, morphologic and functional hematopoietic cells abnormalities, and an increased risk of transformation to acute myeloid leukemia. Previous work from our lab has shown that MDS patients had an impairment in platelet activation, more externalization of phosphatidylserine (PS) indicating an increased platelet apoptosis and, in some cases, thrombocytopenia. However, these patients do not bleed at the frequency that would be expected taking into account these facts. Objective:The aim of this work was to identify the existence of some compensatory mechanisms that could be counteracting the expected bleeding tendency in these patients.Since apoptosis is accompanied by membrane blebbing and production of PS-exposing procoagulant microparticles (MP) shed from the plasma membrane, we assessed the thrombogenic capacity of MP and evaluate their correlation with exposure of PS. We also studied the ability of plasma to generate thrombin after stimulus. Methods:Seventy-five patients with MDS, 36% female, mean age 77 years (range: 41 to 93 years) and sixty-eight healthy controls, 29% female, mean age 52 years (range 20 to 76 years) were included. Whole blood was collected in citrated tubes and centrifuged at 1,500 g for 15 min at 23°C to obtain platelet poor plasma (PPP). PPP was subjected to 2 additional centrifugations at room temperature (first: 15 min at 1,500 g and second: 2 min at 13,000 g) following SSC-ISTH recommendations to obtain platelet free plasma (PFP). MP procoagulant activity was determined in PFP with the ZYMUPHEN MP-Activity kit (HYPHEN BioMed, Neuville sur Oise, France).Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) as described by Hemker at final concentration of 1pM tissue factor and 4 microM phospholipids. Four CAT parameters were recorded: endogenous thrombin potential (ETP, total amount of thrombin generated over time), lag time (time to the beginning of the explosive burst of thrombin generation), time to peak (TTP, time to reach the maximum thrombin concentration) and peak height (maximum thrombin concentration obtained). Apoptosis was determined by assessing the level of PS externalization by the binding of FITC-Annexin V to platelets by flow cytometry. Thrombocytopenia was defined as platelet count less than 50,000 platelets/microliter. Results:Platelet count in MDS patients varied from 4,000 to 478,000 platelets/microliter. We observed an increased MP-associated procoagulant activity in MDS patients with thrombocytopenia compared to controls (p<0.05). This would be related to the increased apoptosis observed in platelets from MDS patients with thrombocytopenia (Spearman test r = 0.655, p <0.01). CAT evaluation of plasma procoagulant activity did not show differences between patients and controls in any of the measured parameters. Conclusions:Our results showed an increased procoagulant activity associated to plasma MPs in MDS with thrombocytopenia that might contribute to the minor risk of bleeding observed in these patients. However, further studies are necessary to assess the clinical significance of this finding. Disclosures:No relevant conflicts of interest to declare.

Lupus anticoagulant hypoprothrombinemia syndrome in Bence-Jones protein κ-type multiple myeloma patient with phosphatidylserine-dependent antiprothrombin antibody

Dear Editor, Approximately 2 % of multiple myeloma (MM) patients present with hemorrhage at diagnosis. However, hemorrhage due to abnormalities in the coagulation system is a rare complication [1, 2]. Although lupus anticoagulant (LA), which is infrequently reported to be in association with MM, is commonly a risk factor for arterial or venous thrombosis, bleeding tendencies in patients with LA are strongly related to a low prothrombin activity [3–6]. Acquired hypoprothrombinemia with LA, also called LA hypoprothrombinemia syndrome (LAHPS), is a rare disease which appears mostly in young females with systemic lupus erythematosus or in healthy children after viral infection and is usually associated with the presence of antiprothrombin antibodies [7]. We herein report the case of an 86-year-old male with Bence-Jones protein (BJP) κ-type MM who presented with hypoprothrombinemia and LA associated with antibodies directed to the phosphatidylserine–prothrombin complex (or phosphatidylserine-dependent antiprothrombin antibodies, aPS/PT). The patient was admitted with anemia and had no past history of bleeding disorders or thrombotic events. A urinalysis showed massive proteinuria (5.3 g/day), which was determined to be κ-type BJP using immunoelectrophoresis. Bone marrow aspiration showed proliferation of abnormal plasma cells. Computerized tomography showed hematomas in the bilateral gluteus maximus muscle and the supraclavicular area. Initial coagulation tests showed prolonged prothrombin time and activated partial thromboplastin time (aPTT) (Table 1). Reduced clotting activity of factors II (FII), VIII (FVIII), and IX (FIX) was noted in a pattern typical of that observed in previously reported cases of LAHPS [8, 9]. FVIII and FIX inhibitors were not detected. The prolonged aPTT with LA-sensitive aPTT reagent (PTT-LA Roche Diagnostics, Tokyo, Japan), which could not be corrected by mixing with normal plasma, suggested the presence of LA. The results were confirmed using the Staclot LA® assay, and the dilute Russell viper venom time test was used to confirm the presence of LA with the phospholipid-neutralizing LA test (Gradipore, Frenchs Forest, Australia). IgG/M anticardiolipin antibodies and IgG aPS/PT were negative, while strong positive IgM aPS/PT was detected, which was measured with ELISA using the phosphatidylserine–prothrombin complex as antigen immobilized on ELISA plates in the presence of CaCl2 [10]. Based on these findings, the patient was diagnosed as MM with LAHPS associated with aPS/PT and treated with melphalan and prednisolone (MP) therapy. The FII levels were observed to normalize after one cycle of MP therapy and the patient has remained in remission without any hemorrhage for 10 months. Table 1 Laboratory findings In our case, aPTT continued to be prolonged with reduced levels of FVIII and FIX in spite of normalizing the FII level after therapy. LA and IgM aPS/PT remained positive, although these values were improved, suggesting that the presence of LA might have an influence on coagulation tests after treatment. There are very rare reports showing the presence of aPS/PT in patients with LAHPS [9]. These reports describe the patients as having bleeding tendencies with mildly reduced FII levels, similar to that observed in our patient. However, in previously reported child cases of LAHPS, severe hemorrhage usually occurs when the FII levels are very low (under 10∼15 %). It is possible that other coagulation factors associated with aPS/PT in LAHPS might be present. A diagnosis of LAHPS should always be considered in MM patients with bleeding tendencies associated with LA, and aPS/PT detection should be performed in conjunction with LA tests.

Protein Z Concentrations in Patients With Acute Leukemia

Protein Z (PZ) deficiency may induce bleeding as well as thrombosis. The aim of our study was to estimate the concentration of PZ in patients with acute leukemia. Plasma levels of PZ were determined in 76 patients with newly diagnosed acute leukemia ([AML], n = 50; acute lymphoblastic leukemia [ALL], n = 26) and 62 healthy participants. In the patients, mean plasma concentrations of PZ were statistically lower than in healthy individuals: AML (1.24 ± 0.11 μg/mL vs 1.58 ± 0.05 μg/mL P = .01) and ALL (1.19 ± 0.16 μg/mL vs 1.58 ± 0.05 μg/mL P = .01). Levels of PZ below the fifth percentile (0.873 μg/mL) of normal value distribution in control participants were found in 30% of patients with AML and ALL and in 3% of controls (P < .0001). In this AML subgroup, we found statistically significant correlation between episodes of bleeding and PZ level (P = .01). There was no such correlation in ALL group. The results suggest that PZ can be a cofactor associated with an increased bleeding tendency in patients with AML.

Bleeding tendency in dual antiplatelet therapy with aspirin/clopidogrel: rescue of the template bleeding time in a single-center prospective study

BackgroundPatients with heightened platelet reactivity in response to antiplatelet agents are at an increased risk of recurrent ischemic events. However, there is a lack of diagnostic criteria for increased response to combined aspirin/clopidogrel therapy. The challenge is to identify patients at risk of bleeding. This study sought to characterize bleeding tendency in patients treated with aspirin and clopidogrel.Patients/methodsIn a single-center prospective study, 100 patients under long-term aspirin/clopidogrel treatment, the effect of therapy was assayed by template bleeding time (BT) and the inhibition of platelet aggregation (IPA) by light transmission aggregometry (LTA). Arachidonic acid (0.625 mmol/L) and adenosine diphosphate (ADP; 2, 4, and 8 μmol/L) were used as platelet agonists.ResultsBleeding episodes (28 nuisance, 2 hematuria [1 severe], 1 severe proctorrhagia, 1 severe epistaxis) were significantly more frequent in patients with longer BT. Template BT ≥ 24 min was associated with bleeding episodes (28 of 32). Risk of bleeding increased 17.4% for each 1 min increase in BT. Correlation was found between BT and IPAmax in response to ADP 2 μmol/L but not to ADP 4 or 8 μmol/L.ConclusionIn patients treated with dual aspirin/clopidogrel therapy, nuisance and internal bleeding were significantly associated with template BT and with IPAmax in response to ADP 2 μmol/L but not in response to ADP 4 μmol/L or 8 μmol/L.

Acquired von Willebrand syndrome in patients with extracorporeal life support (ECLS)

Extracorporeal life support (ECLS) is used for patients with refractory heart failure with or without respiratory failure. This temporary support is provided by blood pumps which are connected to large vessels. Bleeding episodes are a typical complication in patients with ECLS. Recently, several studies illustrated that acquired von Willebrand syndrome (AVWS) can contribute to bleeding tendencies in patients with long-term ventricular assist devices (VAD). AVWS is characterized by loss of the high molecular weight (HMW) multimers of von Willebrand factor (VWF) as a result of high shear stress and leads to impaired binding of VWF to platelets and to subendothelial matrix. Since ECLS and VAD share several features, we investigated patients with ECLS for AVWS. We analyzed 32 patients with ECLS and 19 of them without support. To diagnose AVWS, ratios of ristocetin cofactor activity (VWF:RCo) and collagen binding capacity (VWF:CB) to VWF antigen (VWF:Ag) were employed in conjunction with multimeric analysis. Reduced VWF:RCo/VWF:Ag ratios were identified in 28 ECLS patients. Furthermore, VWF:CB/VWF:Ag ratios were decreased in 31 patients. HMW multimers of VWF were missing in the same 31 patients. Thus, 31 of 32 ECLS patients presented with AVWS. Twenty-two of the 32 patients suffered from bleeding complications. Without support, AVWS was not detectable in any analyzed patient. Our data indicate that AVWS is a typical disorder in patients with ECLS. We hypothesize that AVWS could contribute to aggravation of bleeding tendencies in ECLS patients.

Homozygous F5 deep‐intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet‐rich plasma

Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet-rich plasma (PRP). Therefore, we hypothesized that FV-deficient patients with severe bleeding manifestations may lack platelet FV. To characterize a FV-deficient patient with a severe bleeding diathesis. We performed FV mutation screening and functional studies in a 31-year-old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. The proband was homozygous for a deep-intronic mutation (F5 IVS8 +268A→G) causing the inclusion of a pseudo-exon with an in-frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the proband's plasma or platelets with a prothrombinase-based assay. Moreover, no thrombin generation was observed in PRP triggered with 1-50 pm tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the proband's whole blood, as assessed by thromboelastometry, was markedly delayed but not abolished. This is the first report of a pathogenic deep-intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.

Thrombin generation in patients with factor XI deficiency and clinical bleeding risk

Factor XI (FXI) deficiency is a rare bleeding disorder. Most patients with FXI deficiency are mild bleeders but certain patients with similar FXI activity exhibit different bleeding phenotype. Routine laboratory assays do not help physicians to estimate the individual bleeding risk in these patients. Thrombin generation test (TGT) is a more comprehensive, global function test of the clotting system. We investigated whether or not the bleeding tendency of patients with FXI deficiency is correlated with features of the TGT. Twenty-four patients with FXI deficiency were divided in two groups: (i) severe bleeders (n = 9) and (ii) mild or non-bleeders (n = 15). All severe bleeders had a personal history of surgery-related severe bleeding. Thrombin generation (TG) was measured in platelet-rich plasma (PRP) using a low concentration of tissue factor 0.5 pm. In patients exhibiting severe bleeding tendency, independently of their FXI level, a dramatically impaired TG was observed. For example, despite a low plasma FXI = 1 IU dl(-1), a clinically non-bleeding individual exhibited normal TG results whereas another patient with severe bleeding history and FXI = 40 IU dl(-1) had a very low TG capacity. Low velocity and delayed TG were the main parameters suggesting a higher bleeding risk. DNA analysis of patients reported eight novel mutations of the FXI gene but neither mutation location nor secretion or not of the variant correlated with the bleeding tendency. The results of this study suggest that TG measurement in PRP may be a useful tool to predict bleeding risk in FXI deficiency and should be studied further in larger prospective clinical studies.

The working group for “basic science” in ITP at the International Cooperative ITP Study Group (ICIS)

A variety of studies were considered necessary to better understand the etiology of idiopathic thrombocytopenic purpura. Apoptosis of platelets and megakaryocytes were strong proposals, calling for collaboration. Some groups want to study microparticles and thombin generation in order to understand the variation of the bleeding tendency in patients. Viral antigen cross-reaction with platelet epitopes was another topic that was addressed. Kinetics of cytokines may further contribute to our understanding of the disease.

Modified thromboelastography evaluation of platelet dysfunction in patients undergoing coronary artery surgery

Anti-platelet therapy is associated with increased perioperative bleeding. Although current guidelines call for its caessation 5-10 days prior to cardiac surgery, this could constitute an increased risk of preoperative myocardial infarction. The optimal safe period from discontinuation of anti-platelet therapy to surgery is as yet unknown for the individual patient. We investigated whether preoperative thromboelastography (TEG) with platelet mapping could predict bleeding tendency in patients (on recent anti-platelet therapy) undergoing coronary artery bypass grafting (CABG). We prospectively evaluated 59 patients on aspirin and clopidogrel therapy who underwent CABG. Of them, 25 patients received aspirin alone. TEG with platelet mapping was performed immediately prior to surgery in all 59 patients. During the first 24h post-surgery, 9/59 patients bled excessively (1216 + or - 310 ml in excessive bleeding vs 576 + or - 155 ml in non-bleeding patients). Of the patients bled excessively, eight received clopidogrel treatment prior to surgery. However, 26 of the remaining 34 patients receiving clopidogrel did not bleed significantly. Clopidogrel-induced platelet dysfunction diagnosed by platelet mapping discerned between patients who demonstrated excessive bleeding and those who did not (78% - sensitivity, 84% - specificity, p=0.004). Aspirin-induced platelet dysfunction did not reflect a bleeding tendency. Of all patients, 85% did not respond to a standard dose of clopidogrel, whereas 44% did not respond to aspirin. TEG with platelet mapping is able to predict excessive postoperative blood loss among patients who underwent CABG and recent anti-platelet therapy. The prevalence of non-responsiveness to anti-platelet therapy, including clopidogrel, is higher in patients undergoing coronary artery bypass grafting than in the general population. In this study, aspirin-induced platelet dysfunction did not influence postoperative blood loss.

New concepts of coagulation and bleeding in liver disease

The bleeding tendencies of patients with chronic liver disease have challenged clinicians for centuries. In recent decades, modern science has helped in elucidating some of the mechanisms for this coagulopathy, but there is much to be discovered. It is well known that progressive liver disease is associated with increasing disruption in protein synthesis. Indeed, one of the most ubiquitous of serum proteins, albumin, has been intricately linked to prognosis in cirrhosis patients through the Child–Turcotte–Pugh Score [1], and clinicians have known that dwindling albumin levels are predictive of poor prognosis in these patients. However, it is less widely understood that patients with chronic liver disease may have a persistence of some serum proteins due to ineffective protein degradation. This combination of protein deficiency and persistence contributes to the conflicting knowledge base on coagulopathy in liver disease. In the healthy state of hemostasis, the bleeding and clotting tendencies are balanced. In cirrhosis, this balance is clearly achieved most of the time. The key concept is that the balance is tenuous because of decreased reserve, and when disrupted there can be faltering towards bleeding, especially in the cases where mechanical issues are involved, such as procedural or variceal bleeding. However, there are also circumstances that can lead to abnormal clotting in these patients because of flow issues and local persistence of the prothrombotic proteins. It is the understanding and quantification of this tenuous balance between bleeding and clotting that is shaping our current understanding of the coagulopathy of liver disease. The quantification of this imbalance is the key to using this conceptual framework to establish a clinically useful paradigm for treating patients with chronic liver disease. The widely available clinical laboratory tests used to measure coagulopathy were generally developed to measure quantitative deficiencies in platelet counts or procoagulant factors, and were designed to assess congenital factor deficiencies, such as hemophilia and acquired platelet diseases, such as idiopathic thrombocytopenic purpura. Indeed, the international normalized ratio (INR) was first conceived as a means of standardizing treatment with warfarins. Recent commentary by Tripodi et al. [2], in this journal has outlined the difficulty in using traditional laboratory testing to measure the three fundamental coagulation phases in patients with cirrhosis. In primary hemostasis, which fundamentally involves the platelets and tissue factor, there is a near universal quantitative decrease in platelet numbers in cirrhosis patients, but the data supporting a primary platelet functional deficit is less strong. Based on the level of absolute thrombocytopenia commonly seen in advanced liver disease and early studies suggestive of an additional platelet function disorder [3], it was thought that primary hemostasis was markedly reduced in these patients. However, because much of the function of platelets is dependent on the flow conditions within the vessels, in vitro tests are not representative of the true in vivo function of the platelet. Lisman et al. [4], have extensively studied platelets in flow conditions, and find adequate platelet function using serum of cirrhosis patients due to elevated antigen levels of von Willebrand factor and reduced activity of its cleaving protein, ADAMTS13 [5]. This research has brought into question the magnitude of defect in primary hemostasis in P. G. Northup (&) S. H. Caldwell Division of Gastroenterology and Hepatology, University of Virginia, JPA and Lee Street, MSB 2142, P.O. Box 800708, Charlottesville, VA 22908-0708, USA e-mail: pgn5qs@virginia.edu

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency

We identified 3 mutations in the factor V (FV) gene (F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.

Research for bleeding tendency in patients presenting with significant epistaxis

To evaluate the association of inherited coagulopathies and acquired conditions (e.g. hypertension, aspirin use) with emergency department admission due to epistaxis. Patients admitted to the emergency department with epistaxis were included. A questionnaire for personal and family history of any bleeding disorder was used. Physical examination including ear, nose and throat examination was performed. Platelet counts, International Normalized Ratio, activated partial thromboplastin time, factors VIII, IX and XI, von Willebrand factor and ristocetin cofactor activity levels were determined. Nineteen patients were included in the study. Personal history of mucocutaneous bleeding was present in four cases and family history in two cases. Only one case (5%) had a decreased von Willebrand factor level (45%), and also had a personal and family history of bleeding tendency. Ten patients (53%) had a history of aspirin use. Thirteen (68%) patients had hypertensive values on admission. Aspirin use and hypertension were the leading causes of emergency service admission in adults due to epistaxis in this study, although the number of the patients was relatively low. Regarding the low prevalence of inherited coagulopathies, detailed coagulation tests should be reserved for adult patients with positive personal and/or family history of bleeding.

Platelets as Modifiers of Clinical Phenotype in Hemophilia

Platelets occupy a central role in the maintenance of hemostasis by adhering to sites of vascular injury and facilitating thrombin generation, which leads to the formation of a fibrin clot. Patients with hemophilia exhibit defective thrombin generation secondary to reduced plasma factor concentrations, which can lead to excessive and sometimes life-threatening bleeding. Individuals differ greatly with respect to platelet function and platelets from different individuals differ inherently in their ability to enact thrombin generation, the key coagulative process that is deficient in hemophilia. Similarly, some patients with hemophilia seem to bleed less often than others despite exhibiting similar plasma factor levels. The biologic factors that underlie this phenotypic variability remain poorly understood, but evidence is reviewed supporting a role for platelets and platelet-related factors in modifying bleeding tendency in patients with hemophilia and potential directions for further clinical research in this area are discussed.

Thromboelastography in Children with Coagulation Factor Deficiencies.

The thromboelastograph produces a continuous profile of the rheological changes that occur during the process of coagulation using whole blood. This information can be transformed into a dynamic velocity profile of the changes in blood elasticity occurring during clotting. We used the TEG® hemostasis analyzer in patients with hemophilia A or B with and without inhibitors and other coagulation factor deficiencies (OFD), to study the thromboelastographic profiles in these patients.Materials and Methods: 62 children (6 months-19 years old) were enrolled according to IRB regulations. 29 children had severe hemophilia A (SHA), 4 moderate hemophilia A or B (Mod.H), 2 severe factor VII deficiency, 1 combined factor V and VIII deficiency, 1 VWD (type II B), 1 severe factor V deficiency, 1 Severe PAI deficiency, 19 normal controls (NC), and 4 SHA with inhibitors (SHA+I). All patients were studied 72 hours after the last dose of factor. Citrated whole blood was activated using recombinant human tissue factor (Innovin, Dade Behring Inc®) and recalcified using 0.2M CaCl2. In patients with central lines with heparin, a heparinase cup was used. The TEG® was run for ≥ 90 min. CBC with differential was obtained on all subjects.Results: There was no significant difference in the CBC parameters among patients. Analysis of the TEG data revealed the following:Table 1TEG Parameters (mean values)SHA (n=29)Mod.H (n=4)SHA+I (n=4)OFD (n=6)Control(n=19)MTG(mm*100/sec)8.79.61.3917TMG(min)27.516.662.717.58.9R(min)221456157K(min)744142Max.Amplitude, MA (mm)5956125862MTG:Max rate of thrombin generation; TMG: Time to MTG; R: Reaction Time; K: Time to reach an amplitude of 20mm; MA: Max. AmplitudeThe rate of thrombin generation as visualized by plotting the 1st derivative of the TEG course, in patients with SHA without inhibitors, showed that they could be divided into 2 groups based on MTG (</>9). When analysed the 2 groups showed the following characteristics (5 representative curves from each group are shown): [Display omitted] Table 2TEG Parameters (Mean values)MTG < 9 (n=16)MTG > 9 (n=13)p valueMTG(mm*100/sec)5.512.6<0.001TMG (min)33200.009R(min)26160.004K(min)93.40.03MA(mm)56.162.30.01TMA(min)60380.006TMA: Time to MA;13/29 children with SHA had target joints and 69%of patients with target joints had a MTG<9.Conclusions: SHA patients have variable bleeding tendencies as seen by the variation in MTG. A lower MTG is associated with a higher incidence of target joints. This may provide a clue as to which patients may have the greatest benefit from primary prophylaxis. Patients with OFD have a TEG® profile similar to Mod.H patients. SHA+I have poor thrombin generation as seen by a significantly longer TMG and R time (p <0.05), compared to all subjects. The TEG may provide valuable clues to the severity of bleeding tendencies in patients with factor deficiencies. In additional observations (not shown), it appears that the TEG may be used to monitor the response to treatment with factor concentrates and tailor treatment with rFVIIa.

Increased plasma fibrinolytic activity in bleeding gastrointestinal angiodysplasia

Gastrointestinal angiodysplasia is a major cause of recurrent bleeding. Haemostatic abnormalities have been implicated in the haemorrhage from these common vascular lesions but their precise contribution remains to be established. Our aim was to investigate whether bleeding angiodysplasia is associated with any specific coagulation disorder. Clinical features and blood samples were prospectively obtained from 21 patients with bleeding gastrointestinal angiodysplasia 3 months after the last episode of haemorrhage. Plasma levels of von Willebrand factor, D-dimer, plasminogen activator inhibitor type 1 (PAI-1), tissue-plasminogen activator activity, tissue factor pathway inhibitor and activated factor VII (FVIIa-rTF) were measured. A group of 14 patients with bleeding duodenal ulcer were similarly studied as controls. Mean plasma von Willebrand factor levels were higher in angiodysplasia patients (208+/-12%) than in controls (143+/-11%) (P<0.05). D-dimer levels (661+/-80 ng/ml) and tissue-plasminogen activator activity levels (2.04+/-0.14 IU/ml) were also higher than in controls: 395+/-99 ng/ml and 1.6+/-0.1 IU/ml, respectively (P<0.05), whereas levels of PAI-1, FVIIa-rTF and tissue factor pathway inhibitor were similar in both groups. However, PAI-1 levels (31.5+/-11 ng/ml) were lower in high-bleeding-rate angiodysplasia (more than two bleeding episodes/year) than in low-bleeding-rate angiodysplasia (< or = 2 bleeding episodes/year) (PAI-1 47+/-14 ng/ml) (P<0.05). In a multivariate regression analysis, the plasma level of PAI-1 was a predictor of haemorrhage from angiodysplasia (P<0.05). Increased plasma fibrinolytic activity may contribute to bleeding from angiodysplasia. Low plasma PAI-1 levels constitute a risk factor for bleeding tendency in patients with angiodysplasia.

Impedance-Based Whole Blood and Optical-Based Platelet Rich Plasma Aggregation Methods for the Assessment of Celexocib Effects on Platelet Function: A Preliminary Report.

Cyclooxygenase-2 (COX-2) is the inducible cyclooxygenase isoform expressed by cells involved in inflammation. Platelets normally lack COX-2; however, the enzyme is expressed in megakaryocytes and newly formed platelets and may play a role in clinical syndromes associated with high platelet turnover (1, 2). Selective COX-2 inhibitors (coxibs), have become the mainstay for treatment of patients with arthritis and also have been widely used in cancer chemoprevention. Coxibs were found to have a safer GI toxicity profile when compared with classic NSAIDS; however, in some patients they may increase the risk of GI bleeding (3). The underlying mechanism has not yet been elucidated. Moreover, the role of coxibs in hemostatic alterations has been a subject of controversy. The aim of the study was to investigate the possible anti-platelet effects of celecoxib (200 mg/d for 7 days) in 7 healthy volunteers (2 females, 5 males; mean age = 35.7 ± 8; mean platelet count = 230 ± 23; mean Ht = 42.3 ± 6). A very sensitive whole blood impedance platelet aggregation assay, previously unused for this purpose, was employed for the assessment of celecoxib influence on platelet function. The test was performed along with a traditional, optical-based platelet rich plasma (PRP) aggregation method. Arachidonic acid (0.5; 0.25 mM), Collagen (2; 1mg/mL) and ADP (20, 10 mM) were used as platelet agonists in both tests.Celexocib did not affect platelet function in neither whole blood nor in PRP. The preliminary results of this study indicate that the increased bleeding tendency in patients treated with celecoxib is probably not related to platelet dysfunction.AssayArachidonic AcidCollagenADP0.5[mM]0.25[mM]2[μg/mL]1[μg/mL]20[μM]10[μ M]WB Impedance17.5±4.720.9±3.825.4±3.925.9±6.114.3±2.614.1±5Aggregation: Pre-drugPost-drug18±421±527.1±4.724±418.7±6.216.3±7PRP Optical118.1±10.3118.1±6.7101.4±13.7110±2.2104±15108±8.9Aggregation: Pre-drugPost-drug119.6±6.7112.9±10.2105.4±11.5110.4±7.5102.1±18.3107.7±9.6