Gabriela Cesarman-Maus

Molecular mechanisms of fibrinolysis

The molecular mechanisms that finely co‐ordinate fibrin formation and fibrinolysis are now well defined. The structure and function of all major fibrinolytic proteins, which include serine proteases, their inhibitors, activators and receptors, have been characterized. Measurements of real time, dynamic molecular interactions during fibrinolysis of whole blood clots can now be carried out in vitro. The development of gene‐targeted mice deficient in one or more fibrinolytic protein(s) has demonstrated expected and unexpected roles for these proteins in both intravascular and extravascular settings. In addition, genetic analysis of human deficiency syndromes has revealed specific mutations that result in human disorders that are reflective of either fibrinolytic deficiency or excess. Elucidation of the fine control of fibrinolysis under different physiological and pathological haemostatic states will undoubtedly lead to novel therapeutic interventions. Here, we review the fundamental features of intravascular plasmin generation, and consider the major clinical syndromes resulting from abnormalities in fibrinolysis.

The annexin A2/S100A10 system in health and disease: emerging paradigms.

Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the human ANXA2 gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders.

Autoantibodies Against the Fibrinolytic Receptor, Annexin A2, in Cerebral Venous Thrombosis

Background and Purpose— Cerebral venous thrombosis (CVT) may be a manifestation of underlying autoimmune disease. Antibodies against annexin A2 (anti-A2Ab) coincide with antiphospholipid syndrome, in which antiphospholipid antibodies (aPLA) are associated with thrombosis in any vascular bed. Annexin A2, a profibrinolytic receptor and binding site for &bgr;2-glycoprotein-I, the main target for aPLA, is highly expressed on cerebral endothelium. Here we evaluate the prevalence of anti-A2Ab in CVT. Methods— Forty individuals with objectively documented CVT (33 women and 7 men) and 145 healthy controls were prospectively studied for hereditary and acquired prothrombotic risk factors, classical aPLA, and anti-A2Ab. Results— One or more prothrombotic risk factors were found in 85% of CVT subjects, (pregnancy/puerperium in 57.5%, classical aPLA in 22.5%, and hereditary procoagulant risk factors in 17.5%). Anti-A2Ab (titer >3 SD) were significantly more prevalent in patients with CVT (12.5%) than in healthy individuals (2.1%, P<0.01, OR, 5.9). Conclusions— Anti-A2Ab are significantly associated with CVT and may define a subset of individuals with immune-mediated cerebral thrombosis.

Thrombosis in multiple myeloma (MM)

Abstract Thrombosis is a frequent feature in individuals with myeloma, particularly those treated with oral immunomodulatory drugs (IMID) such as thalidomide or lenalidomide concomitantly with anthracyclines or dexamethasone. Up to a third of these individuals may develop venous thrombosis if not given the benefit of prophylaxis. Interestingly, in contrast to individuals with solid tumors in whom thrombosis is a marker of poor prognosis, thrombosis does not impact overall survival in patients with myeloma. This finding suggests that the mechanisms of thrombosis in hematological neoplasms may differ from solid epithelial tumors and that thrombosis in the former may be driven by therapy and not by a procoagulant phenotype of the neoplastic plasma cells. This may also explain why thrombosis in the context of IMID-based therapy may be prevented by the use of prophylactic aspirin. In this text, we review the pathogenesis of thrombosis in myeloma, its relation to different chemotherapeutic regimens and the use of thrombo-prophylaxis.

Absence of tissue factor is characteristic of lymphoid malignancies of both T- and B-cell origin

BACKGROUND Thrombosis is a marker of poor prognosis in individuals with solid tumors. The expression of tissue factor (TF) on the cell surface membrane of malignant cells is a pivotal molecular link between activation of coagulation, angiogenesis, metastasis, aggressive tumor behavior and poor survival. Interestingly, thrombosis is associated with shortened survival in solid, but not in lymphoid neoplasias. OBJECTIVES We sought to study whether the lack of impact of thrombosis on survival in lymphoid neoplasias could be due to a lack of tumor-derived TF expression. METHODS We analyzed TF gene (F3) expression in lymphoid (N=114), myeloid (N=49) and solid tumor (N=856) cell lines using the publicly available dataset from the Broad-Novartis Cancer Cell Line Encyclopedia (http://www.broadinstitute.org/ccle/home), and in 90 patient-derived lymphoma samples. TF protein expression was studied by immunohistochemistry (IHC). RESULTS In sharp contrast to wide F3 expression in solid tumors (74.2%), F3 was absent in all low and high grade T- and B-cell lymphomas, and in most myeloid tumors, except for select acute myeloid leukemias with monocytic component. IHC confirmed the absence of TF protein in all indolent and high-grade B-cell (0/90) and T-cell (0/20) lymphomas, and acute leukemias (0/11). CONCLUSIONS We show that TF in lymphomas does not derive from the malignant cells, since these do not express either F3 or TF protein. Therefore, it is unlikely that thrombosis in patients with lymphoid neoplasms is secondary to tumor-derived tissue factor.

Newcastle Disease Virus: Potential Therapeutic Application for Human and Canine Lymphoma

Research on oncolytic viruses has mostly been directed towards the treatment of solid tumors, which has yielded limited information regarding their activity in hematological cancer. It has also been directed towards the treatment of humans, yet veterinary medicine may also benefit. Several strains of the Newcastle disease virus (NDV) have been used as oncolytics in vitro and in a number of in vivo experiments. We studied the cytolytic effect of NDV-MLS, a low virulence attenuated lentogenic strain, on a human large B-cell lymphoma cell line (SU-DHL-4), as well as on primary canine-derived B-cell lymphoma cells, and compared them to healthy peripheral blood mononuclear cells (PBMC) from both humans and dogs. NDV-MLS reduced cell survival in both human (42% ± 5%) and dog (34% ± 12%) lymphoma cells as compared to untreated controls. No significant effect on PBMC was seen. Cell death involved apoptosis as documented by flow-cytometry. NDV-MLS infections of malignant lymphoma tumors in vivo in dogs were confirmed by electron microscopy. Early (24 h) biodistribution of intravenous injection of 1 × 1012 TCID50 (tissue culture infective dose) in a dog with T-cell lymphoma showed viral localization only in the kidney, the salivary gland, the lung and the stomach by immunohistochemistry and/or endpoint PCR. We conclude that NDV-MLS may be a promising agent for the treatment of lymphomas. Future research is needed to elucidate the optimal therapeutic regimen and establish appropriate biosafety measures.

Personalizing the use of circulating microparticle-Associated tissue factor as a biomarker for recurrent thrombosis in patients with cancer

TO THE EDITOR: We congratulate Khorana et al for their useful description of tissue factor (TF) as a potential biomarker for recurrent venous thromboembolism (VTE) in patients with cancer undergoing anticoagulation for the treatment of deep venous thrombosis and/ or pulmonary embolism. In patients with cancer, microparticleassociated TF may be detected in the circulation. The origin of these TF-bearing microparticles may be the tumor cell membrane and/or activated endothelium, monocytes, or platelets as a result of host response to cancer or its treatment. In the CATCH (Comparison of Acute Treatments in Cancer Hemostasis) study reported by Khorana et al, the results of microparticle-associated TF levels as determined by enzyme-linked immunosorbent assay from patients with solid (89.6%) or hematologic neoplasias (10.4%) are reported together as a single group; however, hematologic neoplasias, which include diverse diseases such as lymphomas, multiple myeloma, and chronic and acute leukemias, differ frommost solid tumors in their lack of TF expression.We have previously shown that Band T-cell lymphomas, multiple myeloma, and lymphoid leukemias lack TF gene (F3) and TF protein expression, and it is only myeloid leukemias with promyelocytic or monocytic components that may express TF. In Hodgkin lymphoma, which is a B cell–derived tumor, it is the rich microenvironment, not the Hodgkin Reed-Sternberg cells, that expresses TF. Figure 1 shows the striking difference in F3 expression in hematologic versus solid tumors. This contrasted sharply with the common expression of F3 in solid tumors of 74.2% (high in 526 tumors [61.5%] and marginal in 109 [12.7%]). It would be both pathophysiologically and clinically relevant to show the results of circulating TF levels and their relevance to recurrent thrombosis, looking separately at solid and hematologic tumors in the CATCH study. It would also be useful to know whether all of the hematologic tumors were lymphomas. This information would allow for a better, tumor type–directed, personalized use of TF as a potential biomarker for recurrent VTE. In our clinical experience (unpublished data), recurrent VTE in patients with lymphoma is often associated with venous compression resulting from tumor-related adenopathy. Interestingly, in the CATCH study, of the 94 patients with hematologic neoplasias, all four (4.3%) with recurrent DVTseemed to have had venous compression (17% of the 23 patients with venous compression–associated DVT). This information supports the idea that unlike in most solid tumors, TF is not what is driving VTE or recurrent VTE in patients with hematologic tumors. Finally, to interpret the meaning of TF levels, it would be clinically important to knowwhether all patients at diagnosis of the initial VTE indeed had active cancer, because only 52.9% were receiving anticancer therapy (it is unclear in how many patients this was adjuvant treatment). Could high TF levels actually be

Annexin checks in – Response to Waisman

tion as verified by circular dichroism and various functional studies. In contrast, we established that S100A10-bound t-PA, plasminogen and plasmin (MacLeod et al, 2003). We are concerned that the annexin A2 used by Hajjar’s group may be denatured as the protein used in these studies was either passively eluted from polyacrylamide gels or expressed as an extended and tagged recombinant protein. The binding of plasminogen to denatured proteins is a well-established phenomenon. Another important observation by Hajjar’s group was that an antibody to annexin A2 blocked plasmin generation by endothelial cells. However, this antibody (Zymed, Burlington, ON, Canada) has not been characterised and a possible effect on S100A10 could not be ruled out. In contrast, Peterson et al (2003) reported that an S100A10-specific antibody totally blocked endothelial cell plasmin production. Recently, Hajjar’s group demonstrated that a homozygous annexin A2-null mice displayed deposition of fibrin in the microvasculature and incomplete clearance of injury-induced arterial thrombi (Ling et al, 2004). Surprisingly, microarray analysis was not reported for these tissues, so it remained to be established if the levels of proteins other than annexin A2 were altered. It is well-established that knockdown of annexin A2 causes a concomitant loss in S100A10 levels. In fact, the finding of a loss of S100A10 in the tissues of the annexin A2 knockout mouse was recently published by Hajjar’s group (He et al, 2005). The data concerning altered S100A10 levels in the tissues of the annexin A2 knockout mice was neither included in the review (Cesarman-Maus & Hajjar, 2005) nor discussed in the annexin A2 knockout mouse paper (Ling et al, 2004). The decreased S100A10 levels in the annexin A2-null mouse make any conclusion about the role of annexin A2 in plasminogen regulation problematical. We have shown that the selective loss of S100A10 from the surface of either HT1080 fibrosarcoma cells or colorectal epithelial cells results in an 80– 90% reduction in cellular plasmin production. Under these conditions, extracellular annexin A2 levels were unaffected (Zhang et al, 2004). It is unclear as to why the available background information about the role of S100A10 in plasminogen regulation or the potential difficulties in interpretation of the data from the annexin A2 knockout mouse were not discussed by CesarmanMaus and Hajjar (2005). As the major controversy revolves around whether annexin A2 binds plasminogen, we would be happy to exchange reagents with Dr Hajjar to settle these issues.