bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Heparin Antibodies in ICU Patients (The HAICU Study).
Authors: Hergenroeder Georgene W. , Francis John L. , Miller Charles C. , Levine Robert L.
Published: April 2020

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an antibody-mediated prothrombotic disorder characterized by absolute or relative thrombocytopenia occurring after exposure to heparin. Although thrombocytopenia in HIT patients usually resolves after discontinuing heparin, the risk for thrombosis persists up to 3 months.

PURPOSE: The HAICU study was conducted to determine the prevalence of heparin-induced antibodies and HIT on admission to the intensive care unit (ICU) and their development within a week of hospitalization.

METHODS: Platelet counts and heparin-platelet factor 4 antibodies (GTI ELISA) were measured on admission and again on day 7 ± 2 or discharge on a convenience sample of ICU patients. Patients with new onset thrombocytopenia (unexplained platelet count <100 x109/L or a 50% decrease in count) or a positive ELISA had confirmatory testing for HIT antibodies using a serotonin release assay. HIT was prospectively defined as positive HIT antibodies by both assays or as HIT antibodies with new onset, unexplained thrombocytopenia.

RESULTS: Over 6 months, 118 patients were enrolled from three ICUs. Subjects had an average ICU LOS of 9 +/− 11 days, hospital LOS of 20 +/− 11 days and Apache II Physiology Score of 14 +/− 7; 59% were male. HIT, as defined above, occurred in 3 (3%) patients on admission. After 7.2 ± 2 days, HIT occurred in an additional 9 patients. Overall, 12 of 118 (10%) ICU patients had HIT at or within a week of admission (Table 1). 5 had documented prior exposure to heparin within 100 days of ICU admission, and 4 received heparin during the period when HIT assays were performed. Cases were distributed throughout the ICU’s, yet relatively more often in the neurotrauma and shock-trauma units than the medical unit.

CONCLUSION: Heparin-induced antibodies and HIT are common in the ICU, occurring in 3% of patients on admission and 10% in the first week. Physicians need to be aware of heparin exposure and the possibility of HIT in this population.

Patients Positive for Heparin Antibodies by Type of ICU and Time Point Unit No. of Patients Percent (%) Day of ICU Admission Day 7 +/− 2 TOTAL Percent Positive per ICU (%) NTICU 56 47.5 1 5 6 11 STICU 39 33 2 3 5 13 MICU 23 19.5 0 1 1 4 TOTAL 118 100 3 9 12 10
rFVIIa Treatment and Efficacy Prediction in a Patient with Hermansky-Pudlak Syndrome Type 1 (HPS-1) Using Global Clotting Tests and Flow Cytometry.
Authors: Wegert Wolfgang , Scholz Thomas , Ehrenforth Silke , Mondorf Wolfgang , Kirchmaier Carl M.
Published: April 2020

The use of hemostatic agents for patients with platelet function related disorders causing impaired clotting is predominantly monitored via clinical parameters to date. For HPS patients, which suffer from a platelet storage pool disease, up to now mainly desmopressin and/or platelet concentrates have been administered as therapeutic agent in case of bleeding or surgery, with variable success. A more recent option is rFVIIa (NovoSeven®, N7), but there are few reports of its use in HPS patients. Before administering, it would be of therapeutical interest to know the maximum efficacy and the patient’s individual response. We investigated the hemostatic efficacy of rFVIIa in a patient with HPS-1 (63 y; 2 mutations: 972delC, T322N), who was undergoing tooth extraction. rFVIIa (90 U/kg body weight bolus) was administered 30 minutes before the procedure. Citrated blood samples were taken before, 15 minutes after rFVIIa administration and after the dental treatment was completed. Platelet rich plasma was generated by centrifugation (750g, 5 min) and the samples assayed using a fluorometric thrombin generation test (TGT), thrombelastography (RoTEG) and flow cytometry (FACS). The TGT was started adding Ca 16,7 mM and fluorogenic substrate. RoTEG was used with Ca, N7, tissue factor separately and combined. From the pre-substitution samples a reference value with rFVIIa, 80 U/ml added in vitro, and a dose-response curve from 0 to 400 U/ml were measured. N7 recovery was evaluated from pre-, post-substitution and the reference values. The TGT showed a presubstitutional residual activity of 50 % of the maximum ETP value, and 100% after substitution. TGT response to N7 was dose-dependent and entered a plateau at 90 % of the value after substitution, from 80 U/ml N7 on. The effect of subsituted rFVIIa persisted 2h after the extraction. Other TGT parameters (peak, time to peak, lag time) changed concordantly. RoTEG changes were most pronounced for Ca 12 mM alone as a trigger, which showed a transient reduction of clotting time and clot formation time after N7 substitution. FACS showed a 2.2fold CD62P increase indicating platelet activation after administration. Altogether, the TGT parameter changes corresponded best to the clinical outcome, as no bleeding events were observed during and 2h after the dental procedure. Thrombin generation may thus be useful to evaluate and predict N7 therapeutic efficacy in HPS patients. More HPS patients have to be treated to confirm these findings.
Human Thrombospondin-1 in Eleven von Willebrand Factor /FVIII Coagulation Factor Concentrates.
Authors: Suiter Tobias M. , Budde Ulrich
Published: April 2020

Von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury and is therefore essential for primary haemostasis. The modulation of VWF multimers size is critical to the control of the hemostatic activity, where the large multimeric forms are most hemostatically active. It has been shown that various proteases, such as Thrombospondin-1 (facilitates reduction of the disufide bonds), are critical in the degradation of VWF multimers. This might be an important aspect for VWF coagulation factor concentrates used in the treatment of von Willebrands disease, best treated with high molecular weight VWF multimers and least content of human TSP-1 (huTSP-1).

Determination of huTSP-1 (ChemiKine EIA Kit der Fa. Chemicon International, USA) were done in 11 commercially available VWF/FVIII-concentrates (3 different batches each) in duplicates. HuTSP-1 content in the VWF/FVIII concentrates varied from 0,08 ug/mL to 12,7 ug/mL (variance in all duplicates less than 15%), as shown in the figure.

The results show a significant difference in huTSP-1 content. VWD patients might receive as much as 250 ug huTSP-1 or even higher during a single treatment course. If the huTSP-1 is still active, requires further investigations.

Figure Figure
Age-Related Reference Values of Molecular Markers in Endothelial Cell Activation, Coagulation and Fibrinolysis in Thai Children Versus Adults.
Authors: Sosothikul Darintr , Lusher Jeanne M.
Published: April 2020

Introduction: The hemostatic system is a developing and changing process relative to age. Although advances in the knowledge of hemostatic mechanisms have led to the development of new methods for measuring peptides or enzyme-inhibitor complexes, there are very limited data concerning normal reference values for these in children.

Objectives: The aim of this study is to distinguish the difference(s) in various endothelial cell activation and hemostatic parameters between children and adults, and to establish the normal range of these parameters in normal children in different age groups.

Materials and Methods: Blood was obtained from 96 normal Thai children and adults whose screening coagulation tests were normal. All children were categorized into 3 age groups: 1–5 yrs, 6–10 yrs, and 11–18 yrs. Endothelial cell activation parameters: von Willebrand Factor Antigen and Activity (VWF:Ag and VWF:Ac) and soluble thrombomodulin (sTM); coagulation parameters: activated Factor VII (FVIIa:C), tissue factor (TF), and Thrombin-Antithrombin Complex (TAT); and fibrinolytic parameters: tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), D-dimer, thrombin activatable fibrinolysis inhibitor (TAFIa) and Protein C activity (PC:Ac) were measured.

Tests Children’s Age Range Adults (Mean ± SD) 1–5 yr (n=19) 6–10 yr (n=26) 11–18 yr (n=25) (n=25) ANOVA *p ≤ 0.05 compared with adults; **p ≤ 0.001 compared with adults VWF:Ag (%) 93.3 ± 25.7 110.3 ± 27.1 98.4 ± 31.0 100.5 ± 25.2 VWF:Ac (%) 72.1 ± 18.9 91.1 ± 20.6 82.0 ± 24.2 84.7 ± 21.8 sTM (ng/mL) 3.9 ± 1.6** 3.1 ± 0.9 3.1 ± 1.3 2.4 ± 1.1 Fibrinogen (mg/dL) 360.7 ± 56.9 384.4 ± 78.9 410.1 ± 109.4 360.7 ± 51.0 FVIIa:C (%) 68.0 ± 23.8 69.9 ± 23.1 75.0 ± 22.9 75.6 ± 19.7 TF (pg/mL) 218.8 ± 57.8** 152.7 ± 38.2 144.5 ± 46.7 126.6 ± 42.2 TAT (ug/L) 4.5 ± 3.3 2.2 ± 0.7 2.4 ± 1.2 3.7 ± 4.9 t-PA (ng/mL) 1.32 ± 0.56 1.19 ± 0.48 1.16 ± 0.28 1.27 ± 0.32 PAI-1 (ng/mL) 22.8 ± 13.2 29.4 ± 14.2* 27.3 ± 18.3 18.8 ± 11.9 D-dimer (mg/L) 0.52 ± 0.42* 0.44 ± 0.46 0.28 ± 0.33 0.21 ± 0.09 TAFIa μg/mL) ( 40.6 ± 9.7 44.4 ± 6.9 46.1 ± 6.1 42.2 ± 5.7 Protein C:Ac (%) 87.2 ± 18.3* 101.7 ± 15.6 101.1 ± 22.5 102.4 ± 17.4

As seen in the Table, children in all age groups showed no significant difference in mean levels of VWF:Ag and Ac, fibrinogen, FVIIa:C, TAT, t-PA and TAFIa compared to adults. However, compared to adults, children aged 1–5 had significantly higher mean values of sTM (p=0.001), TF (p=< 0.001), and D-dimer (p = 0.015) whereas they had significantly lower mean levels of PC:Ac (p=0.023). The mean levels of PAI-1 in children of all groups were high, especially in children in the 6–10 yr age group (p = 0.032). These data indicate a physiologic difference in endothelial cell activation and hemostatic system between children and adults. Our data will serve as a useful reference guide in interpreting test results from children with suspected bleeding disorders.
Plasma Coagulation on Microparticles Is Modulated by Water Activity. Relevance to Inflammation-Mediated Disruption of the Extracellular Matrix.
Authors: McGee Maria P. , Li Lynne , Ollero Jorge
Published: April 2020

The microenvironment of extravascular spaces influences the reactivities of extravasated plasma proteins. During inflammation, the structural and physicochemical characteristics of peri- and extra-vascular spaces can change widely depending on the severity of extracellular matrix disruption. Plasma extravasation after limited vascular injury results in transient changes in water activity (aw) and fibrin deposition.

This work investigates whether changes in aw influence either pro- or anticoagulant pathways assembled on procoagulant microparticles. Based on conclusions from in vivo models indicating that factor Xa (fXa) concentration is the limiting factor in prothrombinase assembly we modeled the extravascular coagulation process in vitro, using plasma as the matrix; controlling aw as the independent variable; and measuring the fXa generation rate as the dependent variable. Initial rates of fXa generation were measured in human plasma (1/100 final dilution) containing procoagulant particles and 200 nM factor X. Water activity levels decreased from 0 to 0.8 atm relative to isosmolar plasma (~7 atm) with polyethylene glycol 8000 (~26 Å radius), while all reactant concentrations remained constant. Procoagulant particles were either recombinant tissue factor (TF) in PC/PS vesicles (at a mol ratio of 1/2666) or microvesicles collected from the monocytoid cell line THP-1 after 24-hour stimulation with endotoxin (1ug/ml). As the reaction environment’s aw progressively decreased, fXa generation rate first increased, and then decreased. Rate/aw profiles followed nearly symmetrical peaks with a maximum at ~0.2 atm and minimum at ~0.7 atm. Similar profiles were obtained with cell-derived procoagulant microvesicles. At each aw level, the fXa generation rate increased exponentially with TF concentration (25–100 pM). Activation and inhibition indices of ~1.5 and ~2.1, calculated as the ratio between the maximal and baseline and the maximal and minimal rates did not change significantly with TF concentration. Rates increased with added factor VIII (~2 units/ml) indicating fIX activation and a contribution of the intrinsic pathway protease to fXa generation. Furthermore, reaction rates changed with hirudin suggesting that thrombin-mediated regulatory loops also affect the rate profiles. Results in this simple model of extravascular coagulation indicate that aw modulates the rate of coagulation reactions stimulating or inhibiting them depending on whether the reaction environment’s aw is high or low. This conclusion in turn predicts that immediately after limited plasma extravasation coagulation rates accelerate with the decrease in aw as it tend to equilibrate with aw in the extravascular spaces. Continued water adsorption by an intact extracellular matrix would equilibrate aw and decrease coagulation rates. However, in a structurally damaged matrix with compromised water sorption ability, rates may remain longer at the higher level.
Variations in Ex-Vivo Assessments of Hemostasis.
Authors: Gabriel Don A. , Skolnick Brett E. , Rojkjaer Lisa Payne , Seremetis Stephanie V.
Published: April 2020

Previous studies have described differences in hemostasis between Caucasians and African-American subjects, suggesting that ethnicity may influence parameters used to monitor hemostasis. During the course of post-hoc analyses of data from an ex-vivo model to evaluate hemostasis in healthy, non-coagulopathic individuals, evidence of ethnic variability was noted. In this study, ex-vivo assessments of hemostasis were conducted using the thromboelastograph assay (TEG), and Hemodyne Hemostasis Analyzer (HHA). These instruments provide an assessment of platelet function and clot formation. All the subjects enrolled in this trial were healthy male volunteers between the ages of 18 and 45 years. Of the 48 subjects enrolled, 34 (71%) were Caucasians and 14 (29%) were African-Americans. Analyses were performed comparing blood loss volume (assessed by systematic recording of filter paper weights), duration of bleeding, TEG and HHA tracings between the two groups. There were no significant differences in blood loss volume and duration. However in the African-American subjects, TEG analyses demonstrated a significantly higher clot growth rate and maximum clot amplitude strength, while HHA analyses demonstrated a significantly higher platelet contractile force. These observations suggest that ethnic differences in coagulation parameters may exist that could influence techniques or instruments used to evaluate hemostasis. These observations require further investigation. In addition, these observation may provide a useful avenue for further investigation of the apparent enhanced coagulability in the African-American population.

Ex-vivo Assessments of Hemostasis African-American Caucasian n=14 n=31 Mean ± SD Mean ± SD p-value NS, Not statistically significant Blood loss volume (g) 2.19 ± 1.16 2.58 ± 1.84 NS Bleeding duration (mins) 21.07 ± 7.52 20.0 ± 7.21 NS TEG results Coagulation onset time (R, mins) 4.93 ± 0.72 5.40 ± 0.89 NS Time to 20 mm clot strength (K, mins) 1.59 ± 0.25 1.95 ± 0.29 0.0004 Clot growth rate (A, degrees) 67.17 ± 3.78 63.15 ± 3.69 0.0017 Max. clot amp. Strength (MA, mm) 61.07 ± 3.89 57.63 ± 3.96 0.0096 HHA results Clot elastic modulus (CEM, Kdynes/cm2) 20.01 ± 5.80 15.87 ± 7.51 NS Platelet contractile force (PCF, Kdynes) 5.50 ± 1.11 4.42 ± 1.44 0.0165
Effect of Recombinant Activated Factor VII (rFVIIa) on Platelet and Clotting Systems in Healthy Volunteers.
Authors: Gabriel Don A. , Skolnick Brett E. , Seremetis Stephanie , Leese Philip , Mathews David
Published: April 2020

The generation of thrombin is a critical step in clot formation and securing hemostasis. Inadequate thrombin generation may result in excessive bleeding, while uncontrolled thrombin production has the potential to induce thromboses. Recombinant FVIIa has been shown to control bleeding in hemophilia patients with inhibitors to coagulation factors VIII and IX, and several other clinical conditions in which life- and limb-threatening bleeding may occur. However, for non-hemophilia patients the dose and dosing regimen still require refinement. The use of ex-vivo assessment systems (Thromboelastograph assay, Hemodyne Hemostasis Analyzer) to generate an in vitro model of hemostasis may be useful to determine the effect of rFVIIa on the dynamics of clot formation, particularly in a patient population where clinical trials are difficult. A series of pilot studies were performed to establish an optimal punch biopsy location and biopsy size to create an effective bleeding site for further study. The current study (48 subjects consented for an IRB approved protocol) examined, in an ascending dose-escalation manner, the effect of an individual dose of rFVIIa on bleeding time, blood loss volume, coagulation parameters and coagulation status in healthy volunteers. All patients underwent three punch biopsies: the first (baseline biopsy) with no treatment, the second and third biopsy with either a low or high rFVIIa dose administered prior to biopsy. The treatment pair sequences were placebo/10, 10/20, 20/40, 40/80, 80/120, and 120/160 μg/kg. Blood samples were drawn 15 minutes pre-, 15 minutes post-, 1 hour and 5 hours post-biopsy to conduct ex-vivo assessments of hemostasis. The results indicate that overall there was a trend towards an increase in platelet contractile force and clot elastic modulus with an apparent maximal effect at 1 hour post-biopsy. At the higher doses this effect, although reduced, does not return to pre-treatment values by 5 hours post-biopsy. For the lower doses these values decreased to levels comparable to those at pre-treatment. These observations are consistent with the reports of a dose-related half-life of rFVIIa. These data imply that the administration of rFVIIa may have an effect in non-coagulopathic individuals. Although the heterogenity observed in this sample of patients may limit interpretations, it is proposed that this ex-vivo model with further refinements may be useful for evaluating the effects of future pro-hemostatic agents.

Figure Figure
Prophylactic Use of rFVIIa in a Young Hemophilia A Patient with Factor VIII Inhibitor.
Authors: Borel-Derlon Annie , Slaoui Mounia , Gautier Philippe , Guillon Patricia
Published: April 2020

The prevention of bleeding by prophylaxis regimen particularly for joint rehabilitation, could be considered a more effective treatment for hemophilia patients. In hemophiliacs with factor VIII inhibitor (F VIII inh) prophylaxis is not generally proposed because the bypassing agents for these patients may be less effective than F VIII concentrates. We report the regimen and results of a 6 months rFVIIa (Recombinant factor VIIa) prophylaxis, in a young hemophilia A patient (4 years old), with F VIII inh and immune tolerance induction (ITI) treatment and compared, with rFVIIa, the on demand treatment results for the 6 months prior to prophylaxis. After 2 years of a high dose regimen ITI, the FVIII inh titer was less than 50 BU and the immune tolerance treatment remains on going. Due to the development of a right knee target joint the rFVIIa prophylaxis was decided as an active rehabilitation approach to prevent the development of chronic arthropathy as well as to improve the quality of life of the child. During the 6 months period, prior to the initiation of rFVIIa prophylaxis 22 bleeds occurred i.e., 9 right knee hemarthrosis and 13 other joint bleedings and hematoma including elbow, wrist, ankle, foot, arm and chest. These bleeds were all treated with rFVIIa with a dose ranging from 100 to 200 μg/kg depending on the severity of the episodes and the duration of treatment ranged from 1 to 8 days. After 6 recurrent right knee hemarthrosis, a lavage of the joint was performed and prophylaxis with rFVIIa was subsequently initiated. A 120 μg/kg rFVIIa injection was performed 3 times a week concomitantly with the ITI treatment infusion and just before the physiotherapy course. During the 6 months of prophylaxis regimen we observed 9 bleeds with 3 major post traumatic bleedings which were treated by one 200 μg/kg/day rFVIIa injection which was resolved in one to three days. This prophylaxis treatment was effective for the arthropathy evolution and permitted the patient to return to school on a regular basis compared to the previous year. The total dose of on demand rFVIIa treatment used before prophylaxis was 458 mg/6 months. This amount decreased by 25% during the six months of prophylaxis with rFVIIa to reach 343 mg. The results of this significant observation led us to conclude that rFVIIa could be effectively used as prophylactic treatment in patients with FVIII inh and administered safely via a portacath device even in cases of high doses, as demonstrated in this young patient. This prevention approach resulted in a decrease of bleeding episodes, injections, and a significant improvement in the quality of life.
A New Formulation of Recombinant Human Factor IX Is Bioequivalent to BeneFIX: A Double-Blind, Randomized, Crossover Pharmacokinetic and Open-Label Safety and Efficacy Study.
Authors: Powell Jerry Sherman , Lambert Thierry , Navarro F. Hernández , Recht Michael , Sullivan Sharon T. , Udata Chandrasekhar , Lilly Leslie C. , Roth David A. ,   The rFIX-R Study Group
Published: April 2020

A new formulation (rFIX-R) of BeneFIX has been developed to reduce the possibility of RBC agglutination, a phenomenon associated with the low ionic strength of the reconstituted drug product. The new formulation contains NaCl to increase the ionic strength and the recombinant factor IX concentration is increased for the 1000 IU strength and a new 2000 IU strength by reconstituting all strengths in 5 mL, facilitating the administration of larger doses in decreased volumes. Thirty-four (34) previously treated (≥150 exposure days [EDs]) males (≥12 years) with moderately severe/severe hemophilia B (FIX:C ≤2%) were enrolled and treated in this 2 part study. In the randomized, double-blind, crossover pharmacokinetic (PK) period of the study, the PK characteristics of rFIX-R were compared with those of the currently available formulation of BeneFIX. Subjects received either rFIX-R or BeneFIX as a 75 IU/kg bolus infusion over 10 minutes after a suitable washout. Blood samples were collected at defined times over 72 hours to measure pre-/post-infusion plasma factor IX activity. Subjects then received the alternate treatment following the same procedure. The mean plasma factor IX activity-versus-time profile after the infusion of rFIX-R was essentially identical to that seen with BeneFIX. From the 26 evaluable subjects, the mean PK parameter estimates of factor IX activity establish bioequivalence of the products since the 90% confidence intervals for rFIX-R to BeneFIX ratios of the geometric means of Cmax and AUCs fell within the bioequivalence interval of 80% to 125%.

Figure Figure

In the open-label treatment period, subjects used rFIX-R for on-demand control of bleeding, routine prophylaxis, and/or surgical prophylaxis for 6- to 12-months to achieve at least 30 EDs. The plasma factor IX activity-versus-time profile was recharacterized after 6 months of rFIX-R treatment and was unchanged compared with the baseline rFIX-R PK profile. Over 1000 infusions (>3,500,000 IU of rFIX-R) have been administered with no serious adverse events related to rFIX-R. No clinically significant adverse events related to rFIX-R have been reported, including FIX inhibitor development, allergic-type manifestations, or thrombotic complications. One subject, with a prior history of frequent RBC agglutination, reported two instances without any associated adverse events. The overall results of this study demonstrate the safety of rFIX-R and the bioequivalence of the new formulation and BeneFIX. Since bioequivalence is the most important surrogate endpoint for efficacy, these data predict a favorable rFIX-R efficacy profile, consistent with that of BeneFIX, but with increased convenience in that larger doses can be administered using decreased volumes relative to BeneFIX.
Compound Heterozygosity for a Novel Mutation Combined with G Insertion in Exon 13 Causes Severe Factor XI Deficiency in Patients of Japanese Origin.
Authors: Ishikawa Nobutsune , Yasunaga Shinichiro , Ohtsubo Motoaki , Takihara Yoshihiro , Sato Takashi , Kobayashi Masao
Published: April 2020

Factor XI (FXI) deficiency is a rare autosomal recessive coagulopathy. However, it is one of the most common inherited disorders among Ashkenazi Jews. FXI deficiency is characterized by undetectable levels of FXI antigen and coagulant activity. Patients with FXI deficiency usually suffered from mild to moderate bleeding manifestations. Up to now, more than 80 gene mutations responsible for FXI deficiency have been reported including three common mutations (Type I, II and III) in Ashkenazi Jews. However, it has been reported that significantly higher frequency of allelic heterogeneity has been observed in different ethnic groups. We have studied the molecular basis of this disease in a Japanese family. Two children with FXI deficiency who are siblings have frequent epistaxis. Blood coagulation tests showed severely prolonged aPTT with normal PT. FXI coagulant activities of both patients were less than 1% activity. Their father and mother had normal aPTT, but the activities of FXI in parents showed 45% and 52% activities, respectively. Prolonged aPTT restored to normal range by the addition of recombinant Factor VIIa (NovoSevenR) dose-dependently, indicating the possible efficacy for the replacement therapy in this disorder. FXI gene mutations were screened by a PCR. We identified a novel mutation, C to G transversion in exon 12 in the FXI gene. The C to G transversion in exon 12 results in a missense mutaion (Q433E). That leads to the disruption of catalytic domain structure of FXI molecule. Another mutation was found in G insertion in exon 13, which was previously reported in only one Japanese patient, causes the frameshift, resulting in substitution of last 105 amino acids (Tyr503-Val607) with 32 abnormal amino acid residues. This change also induces the destruction of the catalytic domain of FXI. Thus, the compound heterozygous novel mutations found in Japanese are not identical to those in Ashkenazi Jews, suggesting that this mutation may not be of the same ancestry.
The Metalloprotease ADAMTS13 Is a Natural Anti-Thrombotic.
Authors: Chauhan Anil K. , Motto David G. , Lamb Colin B. , Dockal Michael , Scheiflinger Friedrich , Ginsburg David , Wagner Denisa D.
Published: April 2020

The metalloprotease ADAMTS13 cleaves highly adhesive ultra large von Willebrand factor (UL-VWF) multimers after their release from the endothelium. UL-VWF is stored in Weibel-Palade bodies of endothelial cells. ADAMTS13 deficiency is linked to a life threatening disorder, thrombotic thrombocytopenic purpura (TTP), which is characterized by VWF and platelet-rich thrombi in the microvasculature, and may lead to organ ischemia, neurological dysfunction and ultimately death. We previously confirmed in vivo that in Adamts13−/− mice and not in wild-type mice “strings of platelets” formed after endothelial activation. The VWF strings remain attached at one end to the endothelium and “waved” the other end in the blood stream. In addition, we showed prolonged adhesion of platelets to secreted VWF on endothelium of Adamts13−/− mice. The possible role of ADAMTS13 in platelet plug formation has not been addressed previously. We investigated thrombosis in venules and arterioles of Adamts13−/− mice by intravital microscopy. Mesenteric microvenules were stimulated with calcium ionophore A23187, a secretagouge of Weibel-Palade bodies. In the Adamts13−/− mice we observed spontaneous thrombus formation in the activated microvenules. Inhibitory antibody to plasma ADAMTS13 induced thrombi formation in the activated microvenules and prolonged adhesion of platelets to secreted VWF on endothelium of wild-type mice. In the ferric chloride arterial injury model we found that the number of fluorescent platelets deposited within 2–3 min on the sub-endothelium after injury was unexpectedly higher in Adamts13−/− mice as compared to Adamts13+/+ mice (P<0.05). In the Adamts13−/− mice, platelet plug grew faster as thrombi >30 um were seen at 6.6±0.9 min compared to 10.8±0.8 min in the Adamts13+/+ mice (P<0.005). The platelet plug reached occlusive size in 10.6±0.7 min in Adamts13−/− mice, whereas in the Adamts13+/+ mice all the vessels were still open at this time. In the Adamts13+/+, the mean vessel occlusion time was 16.7±1.2 min after injury (P<0.0005). Thus ADAMTS13, by cleaving VWF multimers, down regulates both platelet adhesion to exposed subendothelium and thrombus growth. Furthermore, we observed that recombinant human ADAMTS13 inhibited formation of VWF platelet strings in histamine-activated venules and promoted thrombi dissolution in the Adamts13−/− mice. Our findings reveal that ADAMTS13 has natural anti-thrombotic activity and recombinant ADAMTS13 could be used as an anti-thrombotic agent.
Successful Bilateral Cataract and Circumcision Operation Performed in a Hemophilia-A Patient with Inhibitors by Using Recombinant Factor VIIa.
Authors: Kilinc Yurdanur , Sasmaz Ilgen , Leblebistan Goksel , Antmen Bulent , Sizmaz Selcuk , Yagmur Meltem , Cayli Sibel
Published: April 2020

Objective: Prothrombin complex concentrates (PCC), active prothrombin complex concentrates (APCC;FEIBA) or recombinant Factor VIIa (rFVIIa; Novoseven) are safely used for the treatment of hemorrhages in hemophiliac patients with inhibitors or during the interventions performed in these patients. A hemophilia-A patient with inhibitors in whom bilateral cataract operation and circumcision were successfully performed by rFVIIa administration was presented.

Case: The 13 year old male patient had been diagnosed with severe Hemophilia-A by the detection of a FVIII level below 1% in the factor assay performed due to echhymoses in his body when he was 6 months old. The inhibitor level of the patient who developed bilateral cataract during the follow-up period was determined as 2.95 BU; however the cataract operation had to be delayed due to the absence of an adequate increase in his factor VIII level despite the 2000 U factor replacement administered in July 2002. The patient was readmitted to the hospital for operation in October 2002. However, the operation was delayed again since a safe level of factor VIII wasn’t achieved with Factor VIII replacement. The patient was excluded from follow-up for approximately 2 years. In August 2004, the patient was hospitalized due to echhymosis in the scrotum, and his factor VIII level was measured as 1% and the VIII inhibitor level as 88 BU. IV steroid treatment was initiated in the patient at a dose of 2 mg/kg, with the factor VIII replacement intended at a level of 100 U/kg. 2 doses of 90 microgram/kg of rFVIIa were administered at a two-hour interval due to catheterization for plasmapheresis and subsequently, plasmapheresis program was carried out. The inhibitor level was measured as 25.6 BU after the first plasmapheresis. Factor VIII level of the patient whose FVIII inhibitor level declined to 15.4 BU with these treatments, didn’t exceed 3%. 120 microgram/kg rFVIIa was administered to the patient prior to operation and maintained during the 1st and 2nd days of the operation every 2–3 hours at the same dose. At this operation, right eye small incision cataract extraction and posterior chamber intraocular lens implantation were performed under general anesthesia. A month later, cataract operation was performed in the other eye together with the circumcision operation. A 3-grade increase was achieved in visual acuity of both eyes.

Conclusion: Here, the cataract operation together with the circumcision operation performed in a hemophiliac patient with inhibitors was presented, demonstrating that rFVIIa which has been started to be used also in other fields other than hemophilia patients, can be used safely in operations.
FranceCoag Network: A National Multicenter Prospective Cohort for Congenital Bleeding Disorders.
Authors: Fressinaud Edith , Dieval Jocelyne , Gay Valerie , Guerin Viviane , Lambert Thierry , Donadieu Jean , FranceCoag Reseau
Published: April 2020

The FranceCoag Network is a prospective national cohort of patients affected with hemophilia and severe hereditary hemorrhagic diseases except platelet disorders. It has been set up in France since January 2003 as replacement of a previous project only dedicated to hemophilia. The objectives are to know the exhaustive number, type and characteristics of these patients registered in hemophilia centers. Inclusion criteria are: FVIII or IX defect (<30%), severe defect (<10%) in FII, V, VII, X, XI, XIII or FI (< 0.1 g/l) or severe VWD. Once per year centers send datas on severe bleeding episodes, surgery, replacement therapy i.e. type of concentrate, units, place of treatment and main complications (including new inhibitors). These data are collected and monitored by a coordinating center attached to a public health institution (InVS). Direct transmission of datas via a highly protected web site is foreseen for the end of 2005. The follow-up of the study is ensured by a steering committee (regional coordinators, clinicians, committed health institutions, representatives of patients association) periodically extended to scientific experts and other public health agencies. Beside its epidemiologic interest, this cohort will also promote research projects on this population such as Pups cohort (defined as severe hemophilia A or B patients with none or few cumulative exposure days at the enrolment). On July 2005, 3469 patients were included by 37 centers. The diagnosis are hemophilia A n=2594 (75%); hemophilia B n=539 (15%), VWD n=239 (7%), severe defect in other coagulation factor: n= 97 (3%). At their inclusion, 373 patients (11%) were infected by HIV1, 1383 (40%) had a positive HCV serology, while 365 patients (10%) were co infected by HIV1 and HCV. Among this cohort, 45 deaths were observed since 1994, which represent 4.5 deaths per 1 000 years-patients (95% CI: [3.3–6.0]). Deaths were related to AIDS or HCV in 19 cases. Inhibitors was observed in 388 patients (11%) (hemophilia A: n=369, hemophilia B n=15, VWD n=2, FXIII defect n= 1, FV defect n=1). The incidence of inhibitor during the follow-up was 9.4 cases per 1000 person-years.
Potential Role of ADAMTS13/FXI Complexes in the Pathogenesis of End Stage Renal Disease.
Authors: Fareed Jawed , Hoppensteadt D. , Cunanan J. , Patel C. , Maddineni J. , Baltasar F. , Fareed D. , Demir M. , Tobu M. , Iqbal O. , Bansal V. , Greenfield R.
Published: April 2020

ADAMTS13 alternatively known as von Willebrand Factor (vWF) cleaving protease is a Zinc metalloprotease which cleaves macromolecular M-vWF multimers at the Tyr (1605)-Met (1606) bond located in the AT domain of vWF. Down regulation of ADAMTS13 is known to be associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 forms stable complex with factor XI and factor XIa. Measurement of ADAMTS13/FXI complexes in plasma may provide additional information on the pathogenesis of ESRD associated thrombotic complications. ADAMTS13/FXI can be measured using an ELISA based immunoassay. A polyclonal antibody reacts with a peptide in C-terminal region of the ADAMTS13 molecule is quoted on the micro titer plate. ADAMTS13 present in these test samples bind the antibody. After washing the bound ADAMTS13/FXI complexes were quantified. To test the hypothesis that ADAMTS13 levels are altered in ESRD, blood samples from 62 patients with ESRD prior to dialysis session were collected. Control group comprised of 34 normal healthy age match controls. The vWF antigen levels were also measured in both the groups using an ELISA method. The ESRD group exhibited a marked variation in the ADAMTS13/FXI complex levels with a range of 0 to 1100% (mean = 161±315) of the 62 samples. Eleven patients exhibited a greater than 250% where as the 51 patients exhibited less than 100 % complex. The distribution of ADMTS13/FXI complex was bi-modal. In the normals, the ADAMTS13/FXI complex activity ranges from 147–201% with a mean of 169±16. Unlike the ESRD population the normal group exhibited guassian distribution. The vWF antigen levels relatively higher in the ESRD patients (175±30) vs the normal (88±19). These results suggest that ADAMTS13/FXI is dysregulated in ESRD patients. While most of the patients exhibit a marked decrease in ADAMTS13 level, some of these patients exhibit markedly higher levels. Such fluctuation in the ADAMTS13/FXI level may contribute to the potential thrombotic and bleeding complications in ESRD pathogenesis.
Evidence for Control of von Willebrand Factor Multimer Size by Intramolecular Thiol-Disulfide Exchange.
Authors: Ganderton Tim , Chesterman Colin N. , Berndt Michael C. , Hogg Philip
Published: April 2020

Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF is controlled by proteolysis at the Tyr842-Met843 peptide bond by ADAMTS13. It is apparent, however, that VWF multimers are processed to a significant degree in the absence of ADAMTS13 in mice and humans. This finding implies that another mechanism of controlling VWF multimer size must exist. We noticed similarities between the structural control of α-keratin and VWF. Both are multimeric elongated proteins whose substrates are linked by disulfide-bonds and both proteins undergo shear-dependent conformational change. Keratin shape is controlled by thiol-mediated events and we have shown that the free thiol of thrombospondin-1 can facilitate cleavage of the disulfide-bonds that hold VWF multimers together. The structure of α-keratin is controlled by shear-dependent intramolecular thiol-disulfide exchange. Free thiols in keratin from unpaired cysteines exchange with nearby disulfides when the α-keratin fibre is exposed to shear. The thiol-disulfide exchange process continues until the shear across the fibre is resolved. We hypothesized that a similar mechanism may be operating in VWF. Intramolecular thiol-disulfide exchange is dependent on the presence of at least one unpaired cysteine thiol in the protein. Using a biotin-linked maleimde that specifically alkylates free thiols, we have shown that plasma VWF does contain unpaired cysteine residues. VWF was coated onto the interior of glass capillary tubes and exposed to shear using a precision syringe pump. VWF eluted from the tube when shear was stepped, but not when the shear was constant, and the eluted VWF was of smaller multimer size than that bound to the tube. These observations imply that VWF multimer size can be controlled by intramolecular thiol-disulfide exchange. We suggest that this mechanism may be responsible for the processing of VWF multimer size observed in the absence of ADAMTS13.
Formation of Procoagulant Platelet Derived Microparticles through Platelet GPIb-Interaction with Von Willebrand Factor under High Shear Stress.
Authors: Reininger Armin J. , Heijnen Harry F.G. , Schumann Hannah , Schramm Wolfgang , Ruggeri Zaverio M.
Published: April 2020

We describe here novel findings of the mechanism of initial platelet contact with immobilized von Willebrand factor (VWF) under high shear stress and how this leads to the formation of procoagulant platelet derived microparticles. In a parallel plate perfusion chamber whole blood was perfused over multimeric VWF or dimeric VWF A1 domain at shear rates between 2,000 s−1 and 40,000 s−1. Platelet attachment to VWF always occurred through glycoprotein Ibα receptors located in discrete adhesion points (DAPs), i.e. few limited membrane areas of 0.05 to 0.1 μm2 that arrested the platelets on the surface. The ongoing flow translocated such anchored platelets downstream, thus pulling membrane tethers from the intact and unstimulated platelet. Tethers could remain connected with the platelet body or be eventually severed, which occurred preferentially at shear rates above 6,000 s−1. Depending on the length of the severed membrane fragment they represented either isolated tethers or microparticles (arrowheads; see Figure below), the latter defined by a diameter of 50 to 100 nanometers. The shear rate threshold for microparticle formation was between 6,000 s−1 and 10,000 s−1. Immuno-fluorescence and immuno-electron microscopy showed glycoprotein Ibα clustered in DAPs of microparticles and tethers, i.e. the contact sites with the surface immobilized VWF. The microparticles also exhibited tissue factors on their surface and showed significant procoagulant activity measured by thrombin generation. We propose that after GPIbα anchoring to VWF in flowing blood passive mechanical pulling of membrane from platelets may generate platelet derived microparticles that can potentially support thrombogenesis.

Figure Figure
Comparison of Activated Partial Thromboplastin Time Ratios with Ecarin Clotting Time Ratios for Monitoring Direct Thrombin Inhibitor Therapy: In-Vitro Study and Case Study of Lepirudin Therapy.
Authors: Messmore Harry L. , Fabbrini Nancy J. , Badrinath Ketty , Harriman Richard , Iqbal Omar , Bird Mary Lourdes
Published: April 2020

The direct thrombin inhibitors lepirudin and argatroban are widely used to treat heparin induced thrombocytopenia (HIT). It has been suggested that the Ecarin™ (Echis carinatus venom) clotting time may be superior to the activated partial thromboplastin time (APTT) for monioring purposes. We have prepared standard curves for lepirudin (Refludan™) and Argatroban covering therapeutic drug levels and corresponding APTT ratios (clotting time/control clotting time). Ecarin™ clotting time ratios were performed to demonstrate the practical application of these curves in the clinical care of patients. We report the case of an 80 year old man with HIT/HITT syndrome that occurred during therapy for deep vein thrombosis (DVT) with a low molecular weight heparin (LMWH). His initial coagulation studies were abnormal due to warfarin and LMWH therapy. The patient had a moderate impairment of renal function. Lepirudin therapy in a bolus dose of 14 mg (patient weight: 103.0 kg) resulted in supratherapeutic blood levels of drug and hematuria (Platelet count: 〉200 x 103). Dosage adjustment to maintain an APTT ratio of 1.5 for five days caused no hematuria, but thromboembolic complications occurred at that ratio. The in-vitro dose response curve for Lepirudin was compared with the Ecarin™ clotting time (ECT) ratio at those same concentration ranges in the same plasma. For comparison, Argatroban dose response curves in-vitro were made as well. ECT ratios were very similar to the APTT ratios in the patient’s samples. Representative ratios after the initial bolus, during the infusion period of five days and at the termination of that period are shown in the following table:

Comparison of APTT and ECT Ratios APTT Ratio ECT Ratio 1.43 1.07 3.98 3.08 2.91 1.95 2.74 1.95 2.79 1.90 2.58 1.78 2.44 2.42 3.83 4.78

Conclusion: The ECT ratios reflect a steeper dose response curve than that observed with the APTT ratios. This may permit more accurate measurement of blood levels using ECT ratios.
Two Cases of Pediatric Hit Requiring Unusually High Doses of Direct Thrombin Inhibitors: Does a Subset of Such Patients Exist?.
Authors: Grabowski Eric F. , Buonanno Ferdinando S. , Doody Daniel , Van Cott Elizabeth M. , Grant Patricia E. , Jones Robin M. , Whalen Michael , Nowiski Natan
Published: April 2020

Experience in pediatrics with heparin-induced thrombocytopenia (HIT) with or without thrombosis is currently limited. In the past two years, however, we have cared for two children with HIT who have required unusually high doses of direct thrombin inhibitors in order to maintain a PTT in the 50–80 sec range. Neither child had any recognizeable congenital or acquired risk factor(s) for hypercoagulability, including activated protein C resistance, prothrombin mutation G20210A, deficiency of protein C or S, anticardiolipin antibodies, or elevated levels of homocysteine or lipoprotein (a).

Case 1: A 6 year old girl presented with persistant headache, left-sided weakness, and right lateral gaze paresis. Head CT and MR showed saggital and transverse sinus thrombosis, as well as the presence of two intraparenchymal hemorrhages. Treatment in the first 24 hrs consisted of catheter-directed thromborhexis to recanalize the left transverse sinus and prevent the otherwise anticipated development of complete obstruction to drainage from the right cerebral cortex, followed by low-dose unfractionated heparin (10 U/kg/hr) to inactivate thrombus-bound thrombin released acutely by the thromborhexis. While full anticoagulation was sought by hr 48, this was achieved only over several days owing to a high heparin requirement (45 U/kg/hr) to obtain therapeutic heparin levels. Neurologic status improved over the next several days. However, on day 14 our patient suffered acute loss of vision, following by a drop in platelet count and identification of HIT antibody. She was placed on argatroban, but to attain a therapeutic PTT, required an inordinately high dose (15μg/kg/min, vs “usual” dose of 2). When she was switched to r-hirudin, an unusually high dose (0.7 mg/kg/hr, vs “usual” dose of 0.2) was again required.

Case 2: A 2-and-a-half year old girl was admitted for ECMO following development of respiratory failure due to necrotizing strep. pneumoniae. After 5 days on unfractionated heparin, her platelet count dropped, she developed skin necrosis, and HIT antibody was identified. She was placed on r-hirudin, but also required an unusually high dose (0.52 mg/kg/hr) to achieve a therapeutic PTT. We are aware of two other pediatric cases of such high requirements for direct thrombin inhibitors at other institutions, although a review of the literature makes clear that most instances of pediatric HIT are managed satisfactorily with doses/kg consistent with adult practice. We hypothesize that a subset of chidren with HIT require a significantly higher dose of direct thrombin inhibitors on the basis of a relatively enhanced ability to generate thrombin in the presence of HIT antibody. Studies of thrombin generation and inhibition in our two patients are in progress.