bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Thrombin Production from Single Donor Plasma Using Thrombin Processing Device -TPD™ .
Authors: Kumar Vijay , Zhu Haihong , Chapman John R.
Published: April 2020

Introduction: Thrombin is routinely combined in surgical practice with a fibrinogen source to prepare fibrin sealant to promote hemostasis or with platelet concentrates to prepare gels to enhance wound healing. Currently, thrombin is derived primarily from bovine tissue. The purpose of this study was to evaluate the robustness and reproducibility of the TPD, a new handheld sterile disposable designed to generate active human thrombin in less than 30 minutes from autologous or single donor allogeneic plasma. Thrombin is generated in the device from plasma via the activation of the alternative pathway of the coagulation cascade and thrombin inhibitors (e.g., antithrombin III) are concurrently depleted by a modified Cohn fractionation process. By eliminating the risk of the potential for allergic reactions and blood borne pathogen transmission, autologous human thrombin can be considered the safest thrombin for the preparation of fibrin sealant and platelet gels.

Materials and Methods: Active thrombin was prepared by combining 4 ml of thrombin reagent (a mixture of calcium chloride, ethanol and water) and 11 ml of plasma in a reaction chamber containing negatively charged particles (TPD). The mixture was incubated for 25 minutes and then the active thrombin serum harvested. The thrombin was prepared at ambient room temperatures of 18°C or 25°C (n=100/group) to assess robustness.

Results: The thrombin activity (mean ± S.D.) produced at 18°C and 25°C ambient temperature was 101 ± 22 IU/ml (n=100) and 90 ± 26 IU/ml (n=100), respectively. The average volume of thrombin serum harvested from the TPD was 9.2 ml in both groups. The thrombin concentration generated was demonstrated to rapidly (<5 seconds) coagulate fibrinogen concentrates at a 1:1 ratio (volume to volume). The thrombin maintained clotting activity for ≥4 hours storage at room temperature using a tilt clot test with cryoprecipitate as the source of fibrinogen (less than 5 second clot time).

Conclusions: The results demonstrate that the TPD is able to consistently generate high thrombin activity that is stable for ≥ 4 hours. There was no significant difference in thrombin production at the two extremes of the normal ambient temperature (18° C and 25° C) demonstrating the robustness of the device. The device offers a simple and rapid method for preparing active thrombin from autologous or single donor allogeneic human plasma so as to avoid the safety issues associated with bovine thrombin.
Acquired Hemophilia: Single Center Experience with a Standardized Approach.
Authors: Sucker Christoph , Zotz Rainer B. , Scharf Rudiger E. , Gerhardt Andrea
Published: April 2020

Background: Acquired hemophilia is a rare, often life-threatening, bleeding diathesis caused by autoantibodies, most commonly directed against factor VIII:C. Several clinical studies have reported mortality rates of 16–22 % for affected patients depending on age, inhibitor titre levels, and individual response to treatment. Patients in whom the inhibitor could not be eliminated had a mortality rate of 42 %, providing a strong argument for the necessity of rapid and long-term inhibitor elimination as part of the therapeutic concept. To date, no general consensus exists on the best therapeutic approach.

Patients and Methods: The history and clinical course of ten consecutive patients (4 females, 6 males, mean age [± SD] 61 ± 7 years) diagnosed for acquired hemophilia A in our department are reviewed with special regard to efficacy and side-effects of treatment. Initial treatment consisted of corticosteroids (1 mg/kg) in combination with either azathioprin (1.5 mg/kg, n=7) or cyclophosphamide (1.5 mg/kg, n=3). Severe bleeding complications were treated by recombinant factor VIIa (NovoSeven).

Results: On admission, mean inhibitor titres [± SD] were 83 ± 116 Bethesda units (BU) per milliliter with a range from 10–333 BU. Six of the 10 patients had concomitant disease (chronic obstructive lung disease [n=3], rheumatoid arthritis [n=2], sarcoidosis [n=1]). Immunosuppressive therapy had to be switched from azathioprin to cyclophosphamide in 4 of 7 patients during the clinical course because of treatment-associated hepatitis. Seven of the 10 patients achieved complete remission defined as return to normal factor VIII:C activity and undetectable factor VIII inhibitor titres. Remarkably, the period until complete remission in 2 patients with inhibitor titres above 200 BU was longer (mean 4 months vs. 6 to 8 weeks). Two patients are on immunosuppressive treatment since two months only and experienced a decrease in inhibitor titres, but no increase in factor VIII:C yet. Immunosuppressive therapy was complicated by infection (n=2), development of insulin-dependent diabetes mellitus (n=2), and vertebral fractures (n=1).

Conclusion: Our experience in this limited number of patients demonstrates that immunosuppressive treatment with corticosteroids and cytotoxic agents yields high response rates in acquired hemophiliacs. However, the high frequency of side-effects has to be carefully considered, particularly in elderly patients. In addition, elimination of inhibitors may be delayed specifically in patients with high inhibitor titres, increasing the cumulative risk of bleeding and treatment-associated toxicity.
Association of Acquired Von Willebrand Syndrome with Primary (AL) Amyloidosis.
Authors: Kos Cynthia A. , Ward Jennifer E. , Malek Karim , Sanchorawala Vaishali , Wright Daniel G. , O’Hara Carl , Connors Lawreen , Skinner Martha , Seldin David C.
Published: April 2020

Easy bruising is a common clinical symptom in primary (AL) amyloidosis, and can occur through multiple mechanisms. Infiltration of amyloid fibrils into thin-walled capillaries leads to mechanical fragility predisposing to petechiae and purpura, with periorbital “raccoon-eye” purpura being pathognomonic of AL amyloidosis. Another mechanism predisposing to bleeding in AL amyloidosis is adsorption of coagulation factors to amyloid fibrils. In 1977, Furie et al. reported (New England Journal of Medicine) that elevation of the clotting times is most commonly due to deficiency of factor X, and other investigators have since reported deficiencies of factors II, V, IX, or XIII. We have demonstrated that remission of the underlying plasma cell dyscrasia after high dose melphalan chemotherapy and autologous stem cell transplantation can lead to remission of the acquired factor X deficiency (Choufani et al., Blood 2001). From 2000–2004, four amyloidosis patients presented to Boston University Medical Center with bleeding and a prolonged activated partial thromboplastin time (aPTT), but with no such factor deficiency. Instead, they were found to have abnormal von Willebrand ristocetin cofactor (vWF:RCo) and/or factor VIII (FVIII:C) activities, with normal vWF antigen (vWF:Ag), consistent with a functional defect in von Willebrand factor (vWF). None of the patients had a prior history or family history consistent with congenital von Willebrand’s disease, thus they were diagnosed with acquired von Willebrand syndrome (AvWS). AvWS has most often been reported in association with other lymphoproliferative or myeloproliferative disorders. The aPTT was prolonged in three out of the four cases. Loss of high molecular weight multimers (HMWM) was observed in two of the four cases. Two of the patients were treated with high-dose intravenous melphalan followed by autologous stem cell transplantation and achieved remission of their underlying plasma cell disease; in addition, the bleeding diathesis ceased and the coagulation parameters normalized, indicating reversal of the AvWS with effective treatment of AL amyloidosis.
Improved Coagulation in Bleeding Disorders by NASP Treatment.
Authors: Johnson Kirk W. , Broze George J. , Liu Tongyao
Published: April 2020

Additional therapeutic options are needed for patients with bleeding disorders such as hemophilia A, hemophilia B, and severe von Willebrand disease. A novel treatment approach which may benefit such clotting disorders has been identified that, parodoxically, involves heparin-like sulfated polysaccharides. Select molecules of this broad class are largely devoid of anticoagulant activity and are here denoted Non-Anticoagulant Sulfated Polysaccharides (NASPs). Candidate NASPs were shown in clotting assays to demonstrate at least ten-fold lower anticoagulant activity as compared to heparin. Moreover, a subset of NASPs, including pentosan polysulfate (PPS) and fucoidan, improved (i.e. accelerated) the clotting time of human hemophilia A and hemophilia B plasmas or plasma with reduced FVII levels when tested at concentrations ranging from 4–500 nM in dilute prothrombin time (dPT) assays. A broad window of efficacy was evident with both PPS and fucoidan. A mechanism involving potent blockade of the extrinsic pathway downregulator, Tissue Factor Pathway Inhibitor (TFPI), was implicated from modified dPT clotting tests. Improved hemostasis in vivo was observed in mice with hemophilias A or B following low dose subcutaneous administration of PPS or fucoidan, or a combination of NASP plus factor supplement. Studies in hemophilic dogs are in progress. Accordingly, administration of select NASPs may represent a unique approach for treating multiple bleeding disorders.
Therapy of Hereditary TTP with ADAMTS13 Deficiency - A Case Report and Review of the Literature.
Authors: Huang Jian , Jin Jie , Meng Haitao , Zhou Yuhong , Yu Rongxi , Liu Fang , Ruan Changgen
Published: April 2020

Deficiency of von Willebrand factor (VWF) cleaving protease ADAMTS13 is associated with the development of thrombotic thrombocytopenic purpura (TTP). ADAMTS13 gene mutations cause the hereditary TTP. We describe a patient with relapsing TTP, who failed to achieve sustained remission despite therapies with plasma exchange, steroids, vincristine and ATG. There was lower ADAMTS13 inhibitor titre and lower activity of ADAMTS13 enzyma in the patient’s plasma. Sequence analysis of DNA amplified by polymerase chain reaction showed that the patient was homozygous for two novel missense mutations in TSP1 repeated motif domain of ADAMTS13. In both mutations cytisine were subtituded by thymine. One was a Serine to Leucine substitution in NT 3152, exon21(codon S903L); the other is a Arginine to a Tryptophan substitution in NT3727, exon 25(codon R1094W), while both parents were heterozygous for the same mutation. The patient was dialogue as hereditary TTP. The patient receives 600 to 800 ml plasma transfusion every 3 weeks and now she is very well and works again.

Hereditary TTP with ADAMTS13 deficiency could be treated with the infusion of only a few units of plasma, without plasma exchange. Gene therapy may eventually cure hereditary TTP.
Management of Refractory Hemorrhage in a Kala-Azar Patient with Thrombocytopenia and Coagulopathy by Using Recombinant Factor VIIa (rFVIIa).
Authors: Leblebisatan Goksel , Sasmaz Ilgen , Antmen Bulent , Kilinc Yurdanur , Celik Umit Sizmaz , Mert3 Kurthan , Aksaray Necmi
Published: April 2020

Objective: Platelet transfusion is used for the treatment of acute hemorrhages in diseases other than immunological events, which lead to severe thrombocytopenia. Besides the diseases in which production of platelets is completely terminated in the bone marrow and the platelet requirement is increased, immune thrombocytopenia and alloimmunization may also result in not being able to achieve the desired platelet levels with platelet transfusion, and life threatening hemorrhages. rFVIIa which is used for the management of life threatening hemorrhages in hemophiliac patients with inhibitors and in cases of acquired coagulopathies has also been used successfully in patients with thrombocytopenia. Here we present a case with a diagnosis of kala-azar, where nasal hemorrhage couldn’t be managed with conventional methods and required continuous replacement, and finally treated successfully by using rFVIIa.

Case: At admittance to hospital, hepatosplenomegaly and pancytopenia were detected in the fifteen year old male patient. He was diagnosed with kala-azar upon the detection of Leishmania donovani amastigote form in the examination for bone marrow aspiration. Treatment with sodium stibogluconate was initiated (60 mg/kg). Replacements were performed in the patient due to thrombocytopenia and anemia, who developed nasal hemorrhage during follow-up and had anterior nasal cushion placed. The patient was supplemented with fresh frozen plasma, vitamin K and fibrinogen for his acquired coagulopathy. Since coagulopathy wasn’t corrected and nasal hemorrhage continued actively, 5-minute infusions of rFVIIa were administered to the patient at a dose of 4.8 mg (120 microgram/dl) three times every two hours. His hemorrhage was stopped by rFVIIa treatment.

Conclusion: Our case demonstrated that use of rFVIIa for the management of hemorrhage in thrombocytopenic patients is also feasible in cases other than coagulopathy and that it may be an option for patients with hemorrhages that can not be managed with intensive platelet and plasma transfusions. This result shows that rFVIIa can be used effectively in other diseases other than hematological disorders that cause hemorrhage due to coagulopathy and thrombocytopenia.
Interferon-α-Associated Acquired Factor VIII Inhibitor.
Authors: Hayek Fadi N. , Wagner Stephanie , Laber Damian A. , Sharma Vivek R.
Published: April 2020

Introduction: Development of an acquired inhibitor to factor VIII (acquired hemophilia) is a rare autoimmune phenomenon that has been associated with a variety of medical conditions such as autoimmune diseases, malignancies, drugs like penicillin and postpartum state. It often presents with acute, life-threatening hemorrhage. Interferon alpha (IFN) is an immune system modulator that is widely used for the treatment of hepatitis C and various malignancies. It is known to stimulate or exacerbate autoimmune diseases and has been described in four separate reports to be associated with the development of inhibitors to factor VIII. All but one of these four cases represented patients with underlying malignancy or congenital hemophilia A.

Case Report: Our patient is a 55-year old non-hemophiliac with no prior diagnosis of cancer who developed new-onset bruising while on IFN therapy for hepatitis C. This was not evaluated or treated further at that time. Six months after completing IFN therapy, he presented to the emergency room with respiratory distress. On examination, he was found to have a large supraglottic hematoma and underwent an urgent tracheostomy for impending respiratory failure. This was followed by severe postoperative bleeding which the otolaryngologists were unable to control surgically. Laboratory testing showed a prolonged activated prothrombin time and infusion of fresh frozen plasma failed to achieve hemostasis. A hematology consultation was obtained and the patient was treated with activated recombinant factor VII (rVIIa) which resulted in prompt cessation of bleeding. Existence of factor VIII inhibitor was confirmed by abnormal mixing studies (no correction), low factor VIII activity (2.1%) and high inhibitor titer (42 Bethesda Units/ml [BU]). Following hospital discharge, inhibitor titers slowly declined to less than 1 BU after immunosuppressive therapy with steroids, alkylating agents and rituximab given sequentially over a period of nine months. The factor VIII activity has also increased to 15%. The patient has since had occasional mild to moderate soft tissue bleeding which responded well to rVIIa.

Conclusion: One previous report has described a patient without a history of hemophilia or cancer who developed factor VIII inhibitor while being treated with pegylated IFN for hepatitis C. Our patient represents only the second such report and adds to the slowly growing body of literature pointing to this association. Although this data is anecdotal, we believe that the potential for such inhibitors to produce serious and even life threatening hemorrhage warrants awareness of and attention to this possibility by clinicians treating patients with IFN. Simple clinical vigilance along these lines may help identify and address this condition in a timely fashion before it has a chance to produce catastrophic bleeding.
Technozym® ADAMTS-13 ANTIGEN ELISA - A New Assay for Specific Quantification of the vWF Cleaving Protease ADAMTS-13.
Authors: Vetr Helga , Geiter Sabine , Scheiflinger Fritz , Dockal Michael , Binder Bernd R.
Published: April 2020

Defects in activity and/or antigen levels of ADAMTS-13, the von Willebrand Factor (vWF) cleaving protease, are viewed as the main cause inducing the microvascular thrombotic disorder TTP (thrombotic thrombocytopenic purpura) that is in more than 90% of cases fatal if not treated early and appropriately. Malfunction of ADAMTS-13 with respect to cleave multimeric vWF can be caused by auto-antibodies directed against ADAMTS-13, by decreased presence of ADAMTS-13 in the circulation or by defective activity of ADAMTS-13. Therefore rapid and reliable diagnosis of ADAMTS-13 parameters is a clinical need. We present here an assay for quantification of ADAMTS-13 antigen levels. The assay is a regular double sandwich ELISA employing a monoclonal antibody for capturing ADAMTS-13 from the sample by binding to the CUB domains. The bound ADAMTS-13 is detected by a polyclonal antibody conjugate. The sensitivity of the assay is below 10% of the normal antigen level. In normal samples a wide range (50% to 200%) of the mean level was observed. In idiopathic TTP plasmas also a wide range of antigen levels was observed but with a slighltly lower mean value than in normal samples. Samples from hereditary TTP mostly showed lower antigen levels than normal samples. Thus we could show that this assay provides a useful tool for measuring ADAMTS-13 antigen levels. In combination with our activity assay, which is being developed in parallel, it will be possible to establish ratios of activity and antigen which could provide more insight into the mechanisms of ADAMTS-13 deficiencies than the individual values for antigen and activity.
The C-Terminal α-Helix of von Willebrand Factor Domain A2 Interacts with ADAMTS13 C-Terminal Domains To Regulate Substrate Cleavage.
Authors: Gao Weiqiang , Anderson Patricia J. , Majerus Elaine M. , Tuley Elodee A. , Sadler J. Evan
Published: April 2020

ADAMTS13 is a member of the A Disintergrin And Metalloprotease with ThromboSpondin type I repeat family, and it cleaves the Tyr1605-Met1606 bond in the von Willebrand factor (VWF) central A2 domain, thereby decreasing platelet adhesion mediated by VWF. Recently a minimal substrate for ADAMTS13 was characterized that consists of GST linked to Asp1596-Arg1668 with a C-terminal 6×His tag (VWF73). Further removal of 9 amino acids that comprise a predicted C-terminal α-helix (VWF64) appeared to eliminate cleavage by plasma ADAMTS13, suggesting a critical role for this helix in substrate recognition. We obtained similar results, but VWF64 was cleaved significantly at long reaction times. For example, plasma ADAMTS13 (0.3 nM, one-tenth of normal plasma) cleaved 50% of 65 nM VWF73 in 2 hours and 50% of 62 nM VWF64 in 24 hours. Similar results were obtained in either 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.1 μM ZnCl2, or 5 mM Tris-HCl, pH 8.0, 10 mM BaCl2. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. Using 3 nM enzyme for 30 min, VWF73 was cleaved ~2-fold faster by ADAMTS13 truncated after the spacer domain (MDTCS) than by ADAMTS13 truncated after the cysteine-rich domain (MDTC). Conversely, VWF64 was cleaved ~3-fold faster by MDTC than by MDTCS. Also, in 30 min MDTCS cleaved 70% of VWF73 and <10% of VWF64, whereas MDTC cleaved 40% of VWF73 and 30% of VWF64. ADAMTS13 truncated after the first TSP1 repeat (MDT) or the disintegrin domain (MD) had markedly reduced activity, but with prolonged incubation (24 h) at increased concentration (30 nM) both enzymes cleaved most of VWF64 and VWF73 at the expected site. No aberrant cleavage products were detected by Western blotting. The metalloproteinase domain alone (M) was inactive. The selective effects of deleting the cysteine-rich or spacer domain suggest that the C-terminal α-helix of the VWF A2 domain is not essential but facilitates substrate recognition by interacting with specific C-terminal domains of ADAMTS13, particularly the cysteine-rich and spacer domains.
A Case of Prekallikrein Deficiency in a Pediatric Patient.
Authors: Carpenter Shannon L. , Britton Howard A.
Published: April 2020

Prekallikrein (also known as Fletcher factor) is described as one of the contact factors, and when converted to its active form, kallikrein, is responsible for the activation of factor XII and initiation of the intrinsic pathway of the coagulation cascade. Kallikrein also converts high-molecular-weight kininogen to bradykinin, which acts as a potent vasodilator. Deficiency of prekallikrein was first described in 1965 as an autosomal recessive disorder. Hereditary deficiency of prekallikrein is not associated with a clinical bleeding disorder, but does result in a prolonged activated partial thromboplastin time (aPTT). Most cases of prekallikrein deficiency are identified through routine screening. We present a young man who was referred to our Hemophilia and Thrombophilia Treatment Center for a significantly prolonged aPTT and subsequently found to have prekallikrein deficiency. NP is a 7 year-old boy who was found to have an aPTT of 209.6 seconds on pre-operative evaluation for PE tubes and adenoidectomy. Prothrombin time (PT) was normal at 14.7 seconds, with an INR of 1.2. There was no personal or family history of bleeding. Past medical history did include bipolar disorder and attention deficit disorder, for which the patient was taking olanzapine and depakote. A 1:1 mixing study with normal plasma led to complete correction of the aPTT to 28.9 sesconds. Levels of factors VIII, IX, XI, and XII were normal at 98%, 92%, 99% and 129%, respectively. High molecular weight kininogen level was also normal. Thrombin time was normal at 17.1 sec. An incubated aPTT was performed over 3 hours with decrease in the aPTT from 179 seconds to 72 seconds, a phenomenon that has been reported previously, and is thought to be due to auto-activation of factor XII. Prekallikrein was measured at less that 15% (normal reference 55–207%). The patient subsequently underwent adeno-tonsillectomy without any excessive bleeding. This case serves as another example of the benign causes for a prolonged aPTT in the pre-operative setting in addition to those such as lupus anticoagulant or factor XII deficiency. Deficiency of prekallikrein should be included in the differential diagnosis of a prolonged aPTT, particularly in the absence of bleeding symptoms.
Acquired Hemophilia A: A Single Institution’s Experience.
Authors: Randhawa Devinderpal , Sidhom Ibrahim , Guron Gunwant , Layne Trevor
Published: April 2020

Background: Acquired Hemophilia A (AH) due to factor VIII inhibitor is a rare life threatening disorder. If not diagnosed and treated urgently, significant mortality and morbidity results. AH can occur in setting of old age, autoimmune diseases, pregnancy, medication, malignancy, and lymphoproliferatve disorders. In majority of cases it is idiopathic.

Objective: Review the treatment modalities and outcome of AH patients at our institution.

Methods: A retrospective review of the data pertaining to patients who were diagnosed with AH at our institution between 1993–2004.

Results: There were 5 patients diagnosed with AH, 3 female and 2 male. The median age was 67 years (range 30–84 years) the setting for development of AH in these patients was as follows: 1- postpartum, 1-HIV, 3 idiopathic. All patients presented with varying degree of spontaneous hemorrhage. The median Factor VIII inhibitor level was 16 Bethesda Unit (BU)(range 7. 2–31). Acute control of hemorrhage was achieved in all patients using either FEIBA (Factor eight inhibitor bypass activity) alone (1 patient), FEIBA and Novo seven (VIIa)(4 patients). Chronic immunosuppressive therapy was given as follows: Steroid alone (2 patients), Steroid and IVIG (1 patients), Steroid and Cyclophospamide (1 patient) and Steroid, Cyclophospamide and Rituximab (1 patient). Complete remission (CR) was obtained in 4 patients and with the final patient still receiving treatment. In one patient, the dose of Cyclophospamide was decreased due to Leucopenia. The median time to elimination of inhibitors was 5 month (range 1–10 month). There have been no mortalities.

Conclusions: AH is a life threatening condition if not promptly diagnosed and treated, mortality remains significantly high. Treatment with factors replacement and immunosuppressive therapy was effective in all our patients
Venous Thromboembolism (VTE) in Hematologic Malignancies(HM).
Authors: Korin Jorge D. , Tartas Norma E. , Ferro Hugo , Barazzutti Lucia M. , Kordich Lucia C. , Porterie Pablo , Avalos Julio Cesar Sanchez
Published: April 2020

In our series of consecutive patients (p) with objectively diagnosed VTE, cancer constitutes the most frequent ethiology (25%) followed by idiopathic (24%), clinical causes (22%), orthopedics and trauma (16%), general surgery (7%), obstetric entities (3%) and others (3%). Among the neoplastic subtypes, HM are the most prevalent ones followed by prostatic, colonic, lung and CNS cancers. A recent publication (Blom et al, JAMA2005; 293:715) shows this previously undisclosed high tendency of HM for provoking VTE. Our current study describes the experience with this association from 1996 to 2005, excluding central vein catheter thrombosis. Out of 531 p with VTE, 138 have cancer and 34 of them, HM, with the following distribution: plasma cells discrasias (PCD) 9 (4 on treatment with thalidomide), diffuse large cell lymphomas (DLCL) 8, indolent lymphomas (IL) 6, CLL 5, mantle cell lymphoma 1, Burkitt lymphoma 1, RAEB 1, AML 1, myelofibrosis 1, P vera 1. In 25/34 p, the HM was previously known at diagnosis of VTE and mostly active or on treatment. In the other 9 p, the clinical presentation was as an idiopathic VTE, and during follow-up the following HM were disclosed: PCD: 3, CLL: 2, DLCL: 1, IL: 1, myelofibrosis: 1, RAEB: 1. Table 1

Comments:

PCD and DLCL constitute the two HM more frequently associated with VTE. However, the contribution of less agressive neoplasms such as indolent lymphomas and CLL is not negligible As in solid tumors, recurrences are high, specially when anticoagulated patients are off-therapy In spite of more chemotherapy related-thrombocytopenia and bone marrow involvement, bleeding rates do not differ of those observed in solid tumors Given the frequent association with VTE, and the probable heterogeneity in the thrombophilic potency of these different entities collectively grouped as HM, prospective multicentric studies are clearly needed to identify groups of patients with HM suitable for primary prophylaxis of VTE. Such studies should also be designed to provide further clue about the use of LMWH instead of oral anticoagulants for secondary prophylaxis in HM.

Table 1. Main clinical findings in patients able to be evaluated during follow-up Events Hematologic Malignancies Solid Neoplasms Recurrences 6/28 (21.4%) 5 off anticoagulation 15/106 (14.1%) 7 off anticoagulation (p=ns) Major bleeding 2/28 (7.1%) 10/106 (9.4%) (p=ns)
Proteinase-Activated Receptor 1, 3 Expression in Primary Human Carcinoma Cells.
Authors: Xie Wan-Zhuo , Huang He , Cai Zhen , Lin Mao-Fang , Gieseler Frank
Published: April 2020

The coagulation system is regularly activated in patients with cancer, especially with lung cancer and gastrointestinal tumors. Up to 30% of these patients suffer from thromboembolic complications. Obviously, activation of thrombin occurs in the natural course of cancer. During tumor progression, many patients develop malignant effusions; treatment is complicated by increased resistance of tumor cells to chemotherapy. Thrombin-mediated activation of cellular receptor(s), such as proteinase-activated receptor-1(PAR-1), plays an important role in tumor growth, local progression, and distant metastasis. Our hypothesis is, that tumor cells that express PAR-1 have a survival benefit if they are activated by thrombin within the malignant effusion. We harvested primary cancer cells from pleural and peritoneal effusions of 110 patients with different carcinomas. In 22 cases we were able to establish long time cultures (2 pancreatic, 4 colon, 3 SCLC, 5 NSCLC, 3 mesothelioma, 1 gastric, 1 rectum, 1 breast, 1 melanoma and 1 NHL). We detected PAR-1, 3 expression using reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence activated cell sorting (FACS) scan. We found that in 76% the primary cancer cells were PAR-1 positive using RT-PCR assays, and in 70% the expression of PAR-1 receptor was positive using FACS scan. In contrast, we found that PCR for PAR-3 was positive in only 37.5% and the expression of the receptor in 6%. As a sign of thrombin activation, we regularly found increased levels of prothrombin fragment F1+2 in the malignant effusions compared to serum of patients (12.0 +/− 4.1 nmol/L vs 1.9 +/− 0.4 nmol/L).

In summary, we found that most of the primary cancer cells in malignant effusions exhibit a positive PAR-1 expression, but PAR-3 expression is low. Also, thrombin is activated in most malignant effusions as shown by prothrombin fragment 1+2 elevation. Further experiments will show if these cell-biological mechanisms aggravate cellular resistance in primary tumor cells found in malignant effusion in end-stage cancer. Therapeutic consequences, such as therapeutic thrombin inhibition in addition to chemotherapy are discussed.
High Prevalence of Antibodies to the Heparin-Platelet Factor 4 Complex in Hemodialysis Patients.
Authors: Carrier Marc , Knoll Greg , Kovacs Michael , Rodger Marc A.
Published: April 2020

Heparin-induced thrombocytopenia is a serious complication of systemic heparin therapy. Patients with this disorder develop antibodies to the heparin-platelet factor 4 (H-PF4) complex. Patients with H-PF4 antibodies are at higher risk of venous and arterial thrombosis independently of the platelet count. Hemodialysis (HD) patients are repeatedly exposed to heparin to prevent thrombosis of the extra-corporeal circuit. However, thrombosis remains a frequent complication of vascular access, which accounts for up to 20% of all hospitalization in HD patients. Studies to date on H-PF4 antibody and vascular access thrombosis in HD patients have produced conflicting data but have been limited by small sample sizes. We sough to determine the prevalence of H-PF4 antibodies in a large cohort of HD patients and the correlation with vascular access thrombosis.

Pre-dialysis blood samples were drawn on 419 HD patients; 107 cases with access thrombosis and 312 controls that never had access thrombosis. H-PF4 antibodies were measured twice using an ELISA (GTI PF4 Enhanced, GTI Diagnostics). The average of the two measurements was used in this analysis. All patients received unfractionated heparin while on dialysis. The mean age was 64 16 yrs with 62% male patients. Antibodies to H-PF4 were positive in 54 (12.9%) patients. H-PF4 antibodies were present in 10 (10.5%) of patients with confirmed access thrombosis and in 44 (15.1%) of controls. Hemodialysis with repeated exposure to systemic unfractionated heparin is associated with a high prevalence of H-PF4 antibodies. Although H-PF4 antibodies contribute to hypercoagulability leading to thrombotic complications, our results are not supporting a correlation between H-PF4 antibodies and vascular access thrombosis. Given the potential for venous and arterial thrombosis and the implications of our findings further investigations are needed in this population.
A Case Illustrating That Cancer Tips the Scale of Thrombosis.
Authors: Scott Emma C. , Cohn Jeffrey , Heitner Stephen
Published: April 2020

Background: There are many causes of thrombosis in cancer, including cancer itself, the release of TNF, IL-1 &IL-6 causing endothelial damage, the interaction between tumor cells and macrophages activates platelets, factors X11 &X. Cysteine proteases &tissue factor, in the tumor cells have pro-coagulant activity (Bick, R. New Engl J of Med. Volume 349. July 2003). Chemotherapy, hormonal therapy and indwelling central venous catheters also increase the risk of thrombosis. There is a RR of 4.1 for patients with cancer who did not have chemotherapy and 6.5 for patients with cancer who had chemotherapy (Heit et al. Arch Intern Med. 2000; 160: 809–815).

Factor V Leiden mutation causes partial resistance to the inactivating effects of activated protein C. 5% of the population carries this mutation and it is present in 20% of patients with a 1st venous thrombotic episode. The risk of venous thrombosis is 3–8 fold. The prothrombin mutation is less common and the relative risk of thrombosis is about 2.0. In a large case control study it was found that carriers of the Factor V Leiden or the prothrombin mutation who also had cancer had an approximately 12–17 times increased risk of thrombosis; compared to an overall 7 times risk in patients with malignancy alone (Blom et al. JAMA. Feb 9, 2005. Vol 293, No 6).

Case history: A 54 year old caucasion female was diagnosed with stage 1 infiltrating ductal carcinoma after palpating a lump in her left breast. The tumor was 1.6 cm, ER negative, PR negative and HER-2 negative. Lumpectomy and axillary node dissection revealed no residual carcinoma in the breast and no involvement of eleven lymph nodes. She completed 4 cycles of Cytoxan and Adriamycin successfully and was to start radiation therapy. 1 week after completing chemotherapy she had a focal seizure with tonicclonic movements of her right arm and no loss of consciousness. An MRI showed cortical infarcts, which were originally thought to be metastatic hemorrhages, in the left parietal and frontal areas and right parietal area. A follow up MRI showed considerable improvement of the cortical lesions, with no evidence of any metastatic cancer. Subsequently, she developed bilateral DVTs for which an IVC filter was placed as it was felt that she was not a candidate for anticoagulation given her recent CNS hemorrhage. She was also found to have bilateral pulmonary emboli on CT scan of the chest. Two other underlying disorders predisposing to thrombosis were found- Factor V Leiden mutation and the prothrombin gene mutation. Further imaging confirmed a thrombotic etiology for her CNS event, and coumadin was started.

Conclusion: This case demonstrates the magnitude of the effect of cancer on thrombosis. This patient had 2 prothrombotic mutations, was a smoker, who had been on the OCP for 10 years prior to menopause, yet had no thrombotic episodes until she developed cancer. Also of interest is that the thrombotic episodes occurred shortly after completion of chemotherapy- another prothrombotic factor.
Genetic Basis of Inherited Thrombophilia in Korea: A Single-Institution Experience.
Authors: Kim Hee-Jin , Kim Duk-Kyung , Bang Sung-Hwan , Kim Jong-Won , Kim Sun-Hee
Published: April 2020

Background: Inherited thrombophilia (HT) is a condition that genetically imposes a risk to develop thrombosis. It has been known that the genetic basis of IT is different between Caucasians and Asians; factor V Leiden mutation (R506Q) and the prothrombin gene mutation G20210A are major causes of HT among Caucasians, while they are extremely rare among Asians. Thus the deficiency of anticoagulant factors such as protein C (PC), protein S (PS), and antithrombin (AT) are believed to play a major role in Asians. Genetic study is important to confirm the diagnosis, since there are many acquired conditions including anticoagulant therapy that may mimic inherited form of deficiency. In this study, consecutive patients with a clinical and laboratory suspicion of IT were recruited for genetic diagnosis at a single institution, Samsung Medical Center, Seoul, Korea.

Materials and Methods: From December 2004 to July 2005, a total of 15 patients had presumptive diagnosis of IT; seven were suspected to have PC deficiency (activity, 43~ 51%), four PS deficiency (free Ag; 12~38%), and four AT deficiency (activity, 63~67%). All exons and their flanking sequences of the PROC gene, PROS1, or SERPINC1 were directly sequenced and analyzed.

Results: All seven patients with PC deficiency were shown to have a mutation in PROC. Of note, four of them had a common missense mutation, and the haplotype analysis using polymorphic markers showed that it is a mutation hot focus rather than a founder effect. Two other mutations were novel. Two of four patients with PS deficiency had a mutation in PROS1, each of which was novel. Three of four AT deficiency had a mutation in SERPINC1; one with a novel splice mutation and two shared a common splice mutation, which has not been reported, either. Overall, nine mutation carriers were detected through family study.

Conclusion: This study revealed the genetic basis, mutation spectrum, and the mutation detection rate of IT in consecutive Korean patients. Since diverse (known or novel) mutations underlie genetic deficiency of anticoagulants in Koreans, genetic studies employing direct sequencing for the whole gene are necessary to confirm the diagnosis, and for further family studies.
Correlation between Antifactor Xa Activity and Thrombin Generation Time, Platelet Contractile Force and Clot Elastic Modulus Following Enoxaparin Exposure in Patients with and without Renal Dysfunction.
Authors: Brophy Donald F. , Martin Erika J. , Gehr Todd W.B. , Best Al M. , Carr Marcus E.
Published: April 2020

Patients with renal dysfunction that receive hemodialysis (HD) are highly sensitive to low molecular weight heparin (LMWH) drugs such as Enoxaparin. There have been numerous reports of hemorrhagic effects with these drugs in this population. Although current guidelines recommend judicious monitoring of antifactor Xa activity in HD patients to prevent adverse events, this parameter is poorly correlated to efficacy and toxicity. Newer, more specific parameters such as thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM) may play a role in monitoring LMWH drugs in high-risk populations such as those with renal dysfunction. To determine the utility of monitoring TGT, PCF and CEM for this purpose, we conducted a prospective clinical trial in 8 non-thrombosed HD and 8 control subjects. All subjects received escalating doses of Enoxaparin 0.25, 0.50 and 1.0 mg/kg subcutaneously during this study. Blood samples were obtained four hours post-dose to capture the peak effect of Enoxaparin on antifactor Xa activity, TGT, PCF and CEM. Pearson’s correlation was used to determine the relationships between antifactor Xa activity and TGT, PCF and CEM at each dose, respectively. Repeated measures analysis of covariance was used to assess for intergroup differences in the slopes of each regression line using the group status and antifactor Xa concentration as the covariates. The correlation coefficient (r), coefficient of determination (r2) and p-value for each parameter in each group are listed in the Tables. The figure illustrates the relationship between antifactor Xa and TGT in both controls and ESRD patients. These findings suggest that TGT, PCF and CEM are highly correlated to antifactor Xa activity in patients with and without renal dysfunction. There were no differences in the slopes of the regression lines between groups. Further studies are needed to determine if TGT, PCF and CEM provide more useful clinical information regarding the level of anticoagulation in high-risk patients receiving low-molecular weight heparin therapy.

TGT vs. Antifactor Xa Activity TGT vs. Antifactor Xa Activity

Correlation Parameters for Antifactor Xa and TGT, PCF and CEM for Control Subjects Model r r2 P-value Antifactor Xa vs. TGT 0.88 0.77 0.001 Antifactor Xa vs.PCF 0.85 0.73 0.0003 Antifactor Xa vs. CEM 0.80 0.63 0.02 Correlation Parameters for Antifactor Xa and TGT, PCF and CEM for HD subjects Model r r2 P-value Antifactor Xa vs. TGT 0.91 0.82 0.0002 Antifactor Xa vs. PCF 0.90 0.80 0.0004 Antifactor Xa vs. CEM 0.90 0.80 0.0005
Comparative Study on the Anticoagulant Effects of Low Molecular Weight Heparins as Measured by Whole Blood Act, Anti-Xa and a Newly Developed Prothrombinase Induced Clotting Time (PiCT) Assay.
Authors: Hoppensteadt D. , Tobu M. , Wahi R. , Iqbal O. , Walenga J. M. , Demir M. , Baltasar F. , Fareed J.
Published: April 2020

Low Molecular Weight Heparins (LMWHs) are currently developed for anticoagulation in various intravenous indications such as DVT treatment and percutaneous interventions (PCI). Traditionally, these drugs are administered in anti-Xa units. The purpose of this study was to compare the relative levels of anticoagulation produced by different LMWHs in whole blood ACT (Hemochron), an amidolytic anti-Xa method and a plasma-based global anticoagulant assay, the PiCT test (Pentapharm, Basel, Switzerland). Various LMWHs such as dalteparin, enoxaparin, reviparin and a synthetic pentasaccharide were supplemented to freshly drawn native blood samples to simulate patient samples containing 1 U/ml anti-Xa levels. Three generic versions of enoxaparin were also included in this study. In addition to the ACT measurement in whole blood, citrated plasma samples were also tested for APTT, Heptest, PiCT, amidolytic anti-Xa and IIa activities, and protease generation assays. All agents produced assay-based effects on different tests, which were not proportional to the adjusted anti-Xa activity. Correlation of the ACT data resulted in a good relationship with PiCT for the commercially available branded products(r2=0.97). ACT and Heptest (r2=0.51), ACT and anti-Xa (r2<0.3), ACT and anti-IIa (r2= 0.93). The correlation of PiCT and Heptest was (r2=0.73) better than ACT and Heptest. However, the generic versions of enoxaparin did not provide similar results. PiCT and Heptest also gave the good correlation. These results clearly suggest that for the global anticoagulant actions of LMWHs, anti-Xa measurements are of limited value and should not be used to asses the anticoagulant efficacy of LMWHs in PCI and other surgical conditions. These differences may be more obvious in the generic versions of LMWHs, which are apparently produced using pharmacopeial specifications. Thus, it is important to include a clot-based method in the cross referencing and standardization of LMWHs.
“M-AAA-Thrombin”, a New Thrombin Mutant with Potent Anticoagulant and Antiplatelet Properties.
Authors: Hosokawa Kazuya , Ohnishi Tomoko , Matsuda Hiroyuki , Kashima Kousuke , Koide Takehiko
Published: April 2020

Thrombosis is a major cause of morbidity and mortality, and thrombin is a major inducer of thrombus formation. Thus several antithrombotic agents targeting thrombin have been developed. We previously reported an anticoagulant and antiplatelet thrombin derivative, ‘M-anhydrothrombin’ prepared by chemical modifications. In this study, we prepared a new thrombin mutant, specificity of which was highly modulated with substantially improved antithrombotic efficacy. The thrombin mutant designated “AAA-Thrombin” in which Lys65, His43 and Ser205 in B-chain have been replaced by Ala revealed higher affinity and specificity for factor VIII with no enzymatic activity. AAA-Thrombin prolonged APTT much more than anhydrothrombin in a dose dependent manner without affecting PT and TT. Platelet aggregation induced by activation of PAR-1 was also effectively suppressed by AAA-Thrombin. “M-AAA-Thrombin” prepared by further chemical modification of carboxyl groups in AAA-Thrombin enhanced its antithrombotic efficacy. M-AAA-Thrombin (250nM) prolonged APTT approx. two times, and suppressed platelet aggregation by PAR-1 activation, while AAA-Thrombin did not at the same concentration. M-AAA-Thrombin also suppressed ristocetin-induced platelet aggregation. In vivo experiments, M-AAA-Thrombin demonstrated significant antithrombotic property in the arterio-venous shunt thrombosis model and the FeCl3-induced carotid artery thrombosis model in guinea pigs. These results indicate that M-AAA-Thrombin would be a candidate for quite an innovative anticoagulant and antiplatelet agent for both arterial and venous thromboses. Further optimization of mutagenesis and modification, in terms of efficacy and safety is in progress in our laboratory.
Existing Warfarin Therapy in Long-Term Care Facilities Maybe Inadequate.
Authors: Motlagh Bahareh , Verhovsek Madeleine M. , Papaioannou Alexandra , Mark Crowther , Dolovich Lisa , Campbell Glenda , Kennedy Courtney , Emili Anna
Published: April 2020

Despite evidence-based guidelines derived from large clinical trials supporting the use of warfarin for stroke prophylaxis, studies in elderly patients have shown that oral anticoagulants are not used optimally. The risk associated with inappropriate use is compounded by the observation that the elderly are at enhanced risk of thromboembolic complications compared with younger atrial fibrillation patients. All patients with atrial fibrillation who do not have a contraindication to warfarin, and who meet inclusion criteria, should be treated with warfarin to achieve a target International Normalized Range (INR) of 2.5 (range 2.0–3.0). INR levels of 2.0–3.0 have been shown to be relatively safe and more efficacious than lower target INR values in all age groups including the elderly. Patients with INR values below this range remain at increased risk of thrombosis, while those with INR values above the given range are at increased risk of bleeding. The primary objective of this study was to determine the achieved intensity of warfarin therapy in a cohort of patients living at long-term care facility. In such facilities optimal anticoagulation should be achievable, since laboratory monitoring, dose adjustment, and compliance can be achieved. In this study, data were collected on physicians’ warfarin prescribing practices as well as INR levels of 108 residents in five long-term care facilities in the Hamilton-Wentworth area over a period of 12 months. In total, 3146 INR values, extending over 28,256 patient-days of monitoring, were analyzed. Indications for warfarin were atrial fibrillation, transient ischemic attack, pulmonary embolus, cardiac valve replacement, myocardial infarction, and deep vein thrombosis. In general, the warfarin dosage was not determined using an established dosing algorithm. Our findings revealed that LTC residents spent approximately 40 percent of the time with INR values below 2.0. We therefore conclude, that the overall quality of anticoagulant therapy in long-term care patients may be inadequate. Our observations suggest that organized dosing algorithms may be of benefit in such settings, however this hypothesis needs to be confirmed in prospective studies. For this purpose we plan to implement a warfarin dosing algorithm in order to determine whether the percentage of time spent within the therapeutic INR range can be improved.