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An understanding of reversal strategies alone is important to safely and effectively care for patients in cases of bleeding or invasive procedures. The recent diversification in the number of licensed anticoagulants makes an understanding of drug-specific reversal strategies essential. Intravenous or oral vitamin K can reverse the effect of vitamin K antagonists (VKAs) within 12 to 48 hours and is indicated for any bleeding or an international normalized ratio >10 or 4.5 to 10 in patients with additional risk factors for bleeding. Furthermore, an additional administration of prothrombin complex concentrate (PCC) may be necessary in cases of major bleeding related to VKA. Protamine (chloride or sulfate) fully reverses the effect of unfractionated heparin and partially in low-molecular-weight heparin. Idarucizumab has been approved for dabigatran reversal, whereas andexanet alfa is approved for the reversal of some oral factor Xa inhibitors (apixaban, rivaroxaban). PCC seems to enhance the haemostatic potential for the reversal of the effect of FXa-inhibitors. So far, there are promising but only limited data on the efficacy of this approach available. Each reversal strategy needs an adequate management beyond the hemostatic treatment (volume replacement, stabilization of homeostasis, e.g., pH and temperature, resumption of anticoagulation after successful treatment of bleeding, etc.) that is crucial for the successful management of acute bleedings, urgent high-risk surgery, thrombolytic therapies or thrombectomies as well as overdosing of anticoagulants.

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Rivaroxaban is an oral, direct Factor Xa inhibitor, approved for the prevention and treatment of several thromboembolic disorders. Rivaroxaban does not require routine coagulation monitoring and has a short half-life. However, confirmation of rivaroxaban levels may be required in circumstances such as life-threatening bleeding or perioperative management. Here, we explore the management strategies in patients receiving rivaroxaban who have a bleeding emergency or require emergency surgery. Rivaroxaban plasma concentrations can be assessed quantitatively using anti-Factor Xa chromogenic assays, or qualitatively using prothrombin time assays (using rivaroxaban-sensitive reagents). In patients receiving long-term rivaroxaban therapy who require elective surgery, discontinuation of rivaroxaban 20-30 hours beforehand is normally sufficient to minimize bleeding risk. For emergency surgery, we advise against prophylactic use of hemostatic blood products, even with high rivaroxaban concentrations. Temporary rivaroxaban discontinuation is recommended if minor bleeding occurs; for severe bleeding, rivaroxaban withdrawal may be necessary, along with compression or appropriate surgical treatment. Supportive measures such as blood product administration might be beneficial. Life-threatening bleeding demands comprehensive hemostasis management, including potential use of agents such as prothrombin complex concentrate. Patients taking rivaroxaban who require emergency care for bleeding or surgery can be managed using established protocols and individualized assessment.

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The use of gene transfer techniques has become of utmost importance both for the analysis of molecular pathways of rheumatic joint destruction and for the evaluation of novel therapeutic concepts to treat rheumatic diseases. However, gene transfer into synovial fibroblasts faces several challenges, which result mainly from the lack of specific surface markers and the low-proliferation rate of these cells. This chapter describes both nonviral and viral strategies of transferring gene constructs into synovial fibroblasts. It focuses on the use of lipofection for the gene transfer of siRNA to synovial fibroblasts and the use of AMAXA-nucleofection for the nonviral transfer of gene expression constructs. In addition, retro- and lentiviral strategies of gene transfer are introduced. Finally, the SCID mouse in vivo model of rheumatoid joint destruction is described as a means of evaluating the effects of gene transfer on the invasiveness of synovial fibroblasts.

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OBJECTIVE: Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. METHODS: Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. RESULTS: MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). CONCLUSION: The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.

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Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease with systemic involvement that affects about 1% of the Western population. The progressive destruction of affected joints is a major characteristic of the disease and distinguishes RA from other acute and chronic arthritides. The etiology of RA is unknown, and a variety of genetic and environmental factors are being discussed as potential causes of the disease. However, in contrast to our incomplete understanding of the etiology, the knowledge about molecular mechanisms leading to joint destruction has advanced considerably over the past years. Thus, a large number of studies have investigated the presence and interplay of several types of cells in rheumatoid synovium, such as lymphocytes, macrophages and fibroblasts. They have led to the understanding that cells in the rheumatoid synovium form a network, which interacts through direct cell-to cell contacts as well as the release of a multitude of cytokines. The use of novel molecular techniques together with the development of new animal models has revised our concept on the pathogenesis of RA and specifically on the role of fibroblasts in initiation and progression of joint destruction. This article will review current data and hypotheses on disease mechanisms by which fibroblasts are involved in the destruction of joints in RA.

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OBJECTIVE: To investigate whether retroviral gene transfer of ribozymes targeting matrix metalloproteinase 1 (MMP-1) inhibits the production of MMP-1 in rheumatoid arthritis synovial fibroblasts (RASFs) and reduces the invasiveness of these cells in vivo. METHODS: MMP-1-specific ribozymes (RzMMP-1) were designed and cloned into the pLNSX retroviral vector. Cleavage of MMP-1 was determined in vitro, and the most effective ribozyme was selected for further investigation. RASFs were transduced with replication-deficient viruses carrying RzMMP-1 or with empty viruses (mock). Quantitative polymerase chain reaction with cleavage site-spanning fluorescent probes was used to measure the levels of MMP-1, MMP-9, and MMP-13 messenger RNA. In addition, protein levels of MMP-1 in cell culture supernatants were determined by enzyme linked immunosorbent assay. The effects of stimulation with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) on the production of MMP-1 were assessed accordingly. The invasiveness of RzMMP-1-transduced, mock-transduced, and untransduced RASFs was analyzed in the SCID mouse in vivo model of RA. RESULTS: Transduction of RASFs with RzMMP-1 significantly decreased the production of MMP-1 in RASFs without affecting other MMPs, such as MMP-9 and MMP-13. RzMMP-1 not only reduced the spontaneous production of MMP-1, but also prevented the LPS- and TNFalpha-induced increase in MMP-1 production. Inhibition of MMP-1 was maintained for at least 2 months and was accompanied by a significant reduction of the invasiveness of RASFs in the SCID mouse model of RA. CONCLUSION: Intracellular expression of ribozymes constitutes a feasible tool for inhibiting the production of matrix-degrading enzymes. Inhibition of MMP-1 alone results in a significant reduction of cartilage invasion by RASFs.

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The selective alpha2-adrenoreceptor antagonist, atipamezole, improves behavioural performance of rats subjected to focal cerebral ischemia. The aim of the present study was to investigate whether the facilitatory effect of atipamezole on behaviour is related to altered neuronal activity in specific brain areas. The right middle cerebral artery of rats was occluded for 120 min using the intraluminal filament method. Starting on day 2 after induction of ischemia, atipamezole (1mg/kg, s.c.) or 0.9% NaCl was administered to ischemic or sham-operated rats once a day 30 min before the limb-placing test. [14C]Deoxyglucose ([14C]DG) uptake was used to measure neuronal activity 30 min after atipamezole or 0.9% NaCl administration on day 6 after ischemia. Ischemia induced a significant decrease in [14C]DG uptake in several cortical areas ipsilateral and contralateral to the lesion, in the ipsilateral thalamus, and bilaterally in the cerebellum and spinal cord. Administration of atipamezole normalised [14C]DG uptake particularly in the cerebellum and spinal cord both in sham-operated and ischemic rats and to a lesser extent in the thalamus in sham-operated rats. The pattern of altered cerebral [14C]DG uptake following alpha2-adrenoceptor blockade suggests that plasticity in the cerebellum and spinal cord contributes to the improved performance of ischemic rats in tests assessing tactile/proprioceptive limb-placing reactions.

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