Tami Livnat

Concomitant infusion of low doses of rFVIIa and FEIBA in haemophilia patients with inhibitors.

Summary.  Patients with severe haemophilia A and an inhibitor may become refractory to FEIBA and/or recombinant factor VIIa (rFVIIa). Sequential therapy with both products has been reported in such patients. In this pilot study, we examined the safety and efficacy of combined rFVIIa and FEIBA therapy in patients with haemophilia A and inhibitors during bleeding episodes. We also tried to evaluate whether thrombin generation (TG), by various mixtures of these agents, can serve as a guide for tailoring therapy. TG was measured in plasma taken from eight haemophilia A patients. Increasing concentrations of rFVIIa, FEIBA or both were added ex vivo to the plasmas, and TG was induced by recalcification. Since low concentrations of rFVIIa and FEIBA had either an additive or a synergistic effect in all patients, the lowest combination, yielding TG comparable or lower than TG achieved with either FEIBA 100 U kg−1 or rFVIIa 160 μg kg−1 alone, was selected for the treatment of bleeding episodes. Five patients with a high titre of an inhibitor (8–1300 BU), including one previously refractory to infusions of rFVIIa at doses up to 400 μg kg−1 X4 daily, were treated with combinations of 30–70 μg kg−1 rFVIIa and 20–30 U kg−1 FEIBA during a total number of 400 bleeding episodes with excellent haemostatic effect. No adverse events and no DIC were observed following these infusions. Concomitant infusion of low‐dose rFVIIa and low‐dose FEIBA, seems to be safe, efficacious and economical in patients refractory to rFVIIa and probably other haemophilia A patients with an inhibitor.

Recombinant activated factor VII and tranexamic acid are haemostatically effective during major surgery in factor XI-deficient patients with inhibitor antibodies

One-third of patients with severe factor XI (FXI) deficiency caused by homozygosity for null alleles develop inhibitor antibodies following exposure to plasma. Haemostasis during surgery is achievable in such patients by recombinant activated factor VII (rFVIIa) at doses used in haemophilia A patients with an inhibitor to FVIII. However, thrombosis has occurred in three of 12 such patients. In this study we discerned whether low-dose rFVIIa would secure haemostasis and cause no thrombosis in patients with severe FXI deficiency and an inhibitor during surgery. In vitro, a very low concentration of rFVIIa (0.24 microg/ml) induced thrombin generation in FXI-deficient plasma quite similarly to 1.9 microg/ml (a concentration that is achieved in patients with haemophilia A and inhibitor after infusion of 80 microg/kg). Based on this finding, a protocol was designed for four patients with severe FXI deficiency and an inhibitor or immunoglobulin A deficiency who underwent five major surgical procedures. This included administration of tranexamic acid from two hours before surgery until seven to 14 days after, and single infusion of low-dose rFVIIa. No excessive bleeding or thrombosis were observed. In conclusion, a single low dose of rFVIIa and tranexamic acid secure normal haemostasis in patients with severe FXI deficiency who can not receive blood products.

Effects of factor VIII inhibitor bypassing activity (FEIBA), recombinant factor VIIa or both on thrombin generation in normal and haemophilia A plasma

Summary.  Factor VIII inhibitor bypass activity (FEIBA) and recombinant factor VIIa (rFVIIa) are the common bypassing agents for treating haemophilia A or haemophilia B patients who developed an inhibitor to factor VIII or IX, respectively. As these preparations differ in their composition and mode of action, combined therapy, either sequential or simultaneous has recently been used for achieving haemostasis during bleeding episodes in patients who became refractory to FEIBA or rFVIIa when each was given alone. In this in vitro study, we show by a sensitive assay of thrombin generation that phospholipids present in FEIBA and other procoagulants contribute to FEIBA’s activity and that exogenous phospholipids are essential for the activity of rFVIIa. We also demonstrate that the combination of FEIBA and rFVIIa has a marked synergistic effect on thrombin generation in plasma of a haemophilia A patient with a high titre of an inhibitor. It is conceivable that simultaneous administration of small doses of FEIBA and rFVIIa may be beneficial in treating haemophilia A patients, with an inhibitor to FVIII, who are resistant to conventional therapy.

Degradation of HIF-1alpha under Hypoxia Combined with Induction of Hsp90 Polyubiquitination in Cancer Cells by Hypericin: a Unique Cancer Therapy

The perihydroxylated perylene quinone hypericin has been reported to possess potent anti-metastatic and antiangiogenic activities, generated by targeting diverse crossroads of cancer-promoting processes via unique mechanisms. Hypericin is the only known exogenous reagent that can induce forced poly-ubiquitination and accelerated degradation of heat shock protein 90 (Hsp90) in cancer cells. Hsp90 client proteins are thereby destabilized and rapidly degraded. Hsp70 client proteins may potentially be also affected via preventing formation of hsp90-hsp70 intermediate complexes. We show here that hypericin also induces enhanced degradation of hypoxia-inducible factor 1α (HIF-1α) in two human tumor cell lines, U87-MG glioblastoma and RCC-C2VHL−/− renal cell carcinoma and in the non-malignant ARPE19 retinal pigment epithelial cell line. The hypericin-accelerated turnover of HIF-1α, the regulatory precursor of the HIF-1 transcription factor which promotes hypoxic stress and angiogenic responses, overcomes the physiologic HIF-1α protein stabilization which occurs in hypoxic cells. The hypericin effect also eliminates the high HIF-1α levels expressed constitutively in the von-Hippel Lindau protein (pVHL)-deficient RCC-C2VHL−/− renal cell carcinoma cell line. Unlike the normal ubiquitin-proteasome pathway-dependent turnover of HIF-α proteins which occurs in normoxia, the hypericin-induced HIF-1α catabolism can occur independently of cellular oxygen levels or pVHL-promoted ubiquitin ligation of HIF-1α. It is mediated by lysosomal cathepsin-B enzymes with cathepsin-B activity being optimized in the cells through hypericin-mediated reduction in intracellular pH. Our findings suggest that hypericin may potentially be useful in preventing growth of tumors in which HIF-1α plays pivotal roles, and in pVHL ablated tumor cells such as renal cell carcinoma through elimination of elevated HIF-1α contents in these cells, scaling down the excessive angiogenesis which characterizes these tumors.

A highly sensitive thrombin generation assay for assessment of recombinant activated factor VII therapy in haemophilia patients with an inhibitor

Bypass agents are the common treatment for haemophilia patients who develop inhibitory antibodies. Laboratory assessment of the efficacy of bypassing agent therapy is a challenge. In the present work we modified the conditions triggering thrombin generation (TG) assay in order to find the most sensitive assay for detection of rFVIIa and its analogue NN1731 in haemophilic plasma. TG was measured in samples of normal plasma, plasma of haemophilia patient with inhibitors, as well as haemophilia induced plasma. Recalcification-induced TG was compared to tissue factor (TF) -induced TG in the presence and absence of rFVIIa and NN1731. Recalcification-induced TG (without TF) in haemophilic plasma yielded baseline flat curves, with increased TG as a consequence of spiking the plasma rFVIIa. Using our system, we observed both dose-dependence and time-dependence of rFVIIa effect on TG. Elevated concentrations of TF mask the difference between rFVIIa-treated and non-treated haemophilic plasma. NN1731 yielded normalisation of recalcification-induced TG curves (without TF) which may reflect high potency. In conclusion, we suggest that triggering TG by recalcification-only may be the most sensitive assay for determining the impact of bypassing agents in haemophilic plasma, and may serve as a caution surrogate safety marker in future studies.

The in-vitro effect of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor on clot formation and susceptibility to tissue plasminogen activator-induced fibrinolysis in hemodilution model.

Patients suffering major traumatic or surgical bleeding are often exposed to hemodilution resulting in dilutional coagulopathy. The aim of this study was to evaluate in vitro the effects of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and resistance to fibrinolysis in hemodilution conditions. Citrated whole blood from 36 healthy volunteers was diluted to 30 and 60% with lactated Ringers solution. Blood samples were subsequently supplemented with fibrinogen, FXIII, TAFI or their combinations. Rotation thromboelastometry (ROTEM) in whole blood and thrombin generation in plasma were performed in the presence of CaCl2 and tissue factor/EXTEM reagent, and fibrinolysis was induced by tissue plasminogen activator (tPA). Hemodilution was expressed by decrease of peak height in thrombin generation and &agr;-angle and maximum clot firmness (MCF) in ROTEM. Fibrinogen, FXIII or TAFI did not correct the decrease in thrombin generation peak height. In ROTEM, spiking of diluted blood with fibrinogen stimulated clot propagation. In tPA-treated blood fibrinogen, FXIII and TAFI increased clot firmness and inhibited fibrinolysis. Stronger protection against fibrinolysis was achieved combining FXIII with TAFI. Hemodilution was associated with inhibition of thrombin generation; however, this effect was not sensitive to blood spiking with fibrinogen, FXIII and TAFI. In ROTEM, these hemostasis agents improved clot strength and decreased clot susceptibility to tPA in nondiluted and to more extent in diluted blood. The maximal protection against fibrinolysis was caused by TAFI. Combining FXIII with TAFI exerted synergistic inhibitory effect on fibrinolysis.

The impact of thrombin generation and rotation thromboelastometry on assessment of severity of factor XI deficiency

The phenotype of bleeding in patients with severe FXI deficiency is unpredictable and unlike other bleeding disorders, it is not directly correlated with levels of FXI. In this study we analyzed whether the global coagulation assays can serve as a clinical tool in predicting bleeding tendency in patients with severe FXI deficiency undergoing surgery, taking into account the large inter-individual variability of FXI levels and genotypes. Thrombin generation (TG) was measured in 39 platelet-poor plasma with or without tissue factor (TF) and in the presence or absence of corn trypsin inhibitor (CTI). Rotation thromboelastometry (ROTEM) was performed with fresh whole blood of 26 patients applying NATEM and INTEM tests. TG induced by recalcification can distinguish between bleeding and non-bleeding patients with severe FXI deficiency particularly among those with FXI activity of 2-20IU/dl. The addition of TF or TF and CTI to the TG assay masked the ability to differentiate between XI activity, genotype as well as bleeding and non-bleeding patients. ROTEM assays failed to distinguish bleeding from non-bleeding patients but could do so between different FXI activity levels and genotypes. In conclusion, in the current study we found a sensitive tool to distinguish between bleeding and non-bleeding patients. In order to recommend TG as a predictive tool for treatment tailoring, a larger patient group is required.

The in vivo effect of fibrinogen and factor XIII on clot formation and fibrinolysis in Glanzmann's thrombasthenia

Glanzmanns thrombasthenia (GT) is characterized by increased bleeding risk. The treatment options in GT are limited. The aim of this study was to test the effect of GT blood supplementation with fibrinogen and factor XIII on thrombin generation, blood clotting, and fibrinolysis. Whole blood samples of GT patients and normal donors treated with eptifibatide (GT model) were subjected to clotting by CaCl2 and tissue factor. Thrombin generation was measured in platelet-rich plasma. Clot formation and tPA-induced fibrinolysis were evaluated in whole blood by rotation thromboelastometry (ROTEM). Blood was supplemented with fibrinogen (3 g/L) and/or FXIII (2 IU/mL). Thrombin generation analysis of blood derived from GT model and GT patients revealed decreased endogenous thrombin potential and peak height and extended lag time compared to control. However, this method was not sensitive to blood spiking with fibrinogen and FXIII. ROTEM revealed lower maximum clot firmness (MCF) and area under curve (AUC) in the blood of GT model and GT patients. In the absence of exogenous tPA, blood spiking with fibrinogen markedly enhanced clot quality while FXIII had no effect. Combination of fibrinogen and FXIII did not add to the effect of fibrinogen. In contrast, by the addition of tPA, both fibrinogen and FXIII separately and, to more extent, in combination enhanced clot quality as well as resistance against tPA-induced fibrinolysis (increasing MCF, AUC, and lysis onset time). In conclusion, fibrinogen and FXIII exerted stimulation of blood clotting and inhibition of fibrinolysis. Treating normal blood with eptifibatide mimics the changes of coagulopathy in GT blood.

Enhancement of Angiogenic Potential of Endothelial Cells by Contact with Retinal Pigment Epithelial Cells in a Model Simulating Pathological Conditions

PURPOSE Choroidal neovascularization (CNV) is the leading cause of vision loss in chorioretinal diseases involving contact between retinal pigment epithelial (RPE) and endothelial cells (ECs). The aim of this study was to investigate changes in the angiogenic potential of ECs induced by RPE-EC interaction in two models of RPE-EC coculture. METHODS RPE and ECs were grown in contact or noncontact coculture. Selection of ECs was achieved using magnetic beads coated with antibodies specific for EC surface proteins. Angiogenesis was assessed by analyzing the expression of EC genes involved in angiogenesis by RT-PCR. Tube formation on Matrigel was used as a functional angiogenesis assay. Expression and activity of matrix metalloproteases (MMPs) were examined by RT-PCR and zymography, respectively. RESULTS Coculture of ECs with RPE in the contact model under normoxic conditions induced markedly upregulated EC mRNA expression of 16 genes involved in positive regulation of angiogenesis. Solo ECs subjected to hypoxia demonstrated upregulated expression of the same 16 genes, including VEGF and HIF1. The EC VEGF level was not affected by coculture with RPE in the noncontact model. ECs demonstrated enhanced tube formation on Matrigel after contact coculture with RPE. EC MMP2 mRNA and activity levels were elevated in contact, but not in noncontact, coculture. CONCLUSIONS Coculture of ECs with RPE under conditions enabling direct EC-RPE contact enhances the proangiogenic potential of ECs under normoxia, to an extent similar to that induced by hypoxia, suggesting that ECs in direct contact with RPE cells might be more prone to pathologic angiogenesis involved in CNV formation.

In vitro evaluation of clot quality and stability in a model of severe thrombocytopenia: effect of fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor.

BACKGROUND The treatment options in severe thrombocytopenia (platelet count ≤20×10(9)/L) are limited. The aim of this study was to investigate ways of improving blood clotting and stability in reconstituted thrombocytopenia. MATERIALS AND METHODS Thrombocytopenia (platelets [16±4]×10(9)/L) was created by differential centrifugation of normal blood followed by reconstitution of whole blood which was subjected to clotting in a rotation thromboelastometer by CaCl2 and tissue factor, and to fibrinolysis by tissue plasminogen activator (tPA). In separate experiments, blood was diluted by 40% with TRIS/saline solution. Blood was treated with fibrinogen (fib), factor XIII (FXIII), and thrombin-activatable fibrinolysis inhibitor (TAFI). RESULTS The maximum clot firmness of thrombocytopenic blood was approximately 2-fold less than that of intact blood. Supplementation of blood with fib and FXIII improved clot formation. In the presence of tPA, among fib, FXIII and TAFI, only fib stimulated clot propagation whereas each of these agents increased clot strength. There was a synergistic effect when fib was added together with FXIII or TAFI. Fibrinolysis was inhibited by TAFI and to a greater extent by TAFI + FXIII. Fourty percent dilution of blood reduced clot strength and increased susceptibility to tPA. Clot strength was increased by the treatments in the following order: fib/FXIII/TAFI > fib/TAFI > fib > TAFI > FXIII. In the presence of tPA, TAFI and FXIII lysed the clots significantly more slowly. This effect was stronger when blood was treated with the combination of fib/FXIII/TAFI. Doubling the fib concentration, alone or together with other agents, did not improve clot strength or stability. DISCUSSION Augmentation of clot formation and anti-fibrinolysis by combining fib, FXIII and TAFI may be beneficial for the treatment of patients with severe thrombocytopenia especially when complicated by haemodilution following introduction of fluids to compensate for massive blood loss.