Blood Coagulation Inducing Synthetic Polymer Hydrogel

Uncontrolled hemorrhaging, or blood loss, accounts for upwards of 3 million deaths each year and is the leading cause of preventable deaths after hospital admission around the world. Biological-based hemostatics are quite effective at controlling blood loss, but prohibitively expensive for people in developing countries where over 90 % of these deaths are occurring. Synthetic-based hemostatics are less expensive, yet not nearly as effective as their biological counterparts. A better understanding of how synthetic materials interact with and affect the body's natural clotting response is vital to the development of future hemostatic material technology which will help millions around the world. Initial in vitro experimentation focused on investigating the key chemical and structural material properties which affect Factor VII (FVII) activation in citrated human plasma. Enzyme-linked assays were utilized to confirm the ability of specifically formulated charged hydrogels to induce FVII activation and provided insight into the critical material parameters involved in this activation. Dynamic mechanical analysis was used to establish a correlation between polymeric microstructure and FVII activation. Experiments utilizing coagulation factor depleted and inhibited plasmas indicated that FVII, FX, FII, and FI are all vital to the process outlining the general mechanism of fibrin formation from the onset of FVII activation. The ability of the polymer to induce fibrin formation in "artificial plasma" explicitly lacking calcium, TF, and platelets suggested that a specifically designed material surface has the capability to substitute for these vital cofactors. Clinical diagnostic experimentation using sheep blood indicated that hydrogels containing higher amounts of electrostatic positive charge and lower cross-link density were able to induce faster, more robust clot formation in the presence of a coagulation cascade activator. Subsequent in vivo animal experimentation clearly demonstrated the ability of such hydrogels to aggregate platelets and erythrocytes promoting the formation of an effective hemostatic seal at the wound site. Moreover, in vivo testing confirmed the viability of such a charged polymer hydrogel to effectively control blood loss in a clinically relevant model

Blood Coagulation Inducing Synthetic Polymer Hydrogel

Uncontrolled hemorrhaging, or blood loss, accounts for upwards of 3 million deaths each year and is the leading cause of preventable deaths after hospital admission around the world. Biological-based hemostatics are quite effective at controlling blood loss, but prohibitively expensive for people in developing countries where over 90 % of these deaths are occurring. Synthetic-based hemostatics are less expensive, yet not nearly as effective as their biological counterparts. A better understanding of how synthetic materials interact with and affect the body's natural clotting response is vital to the development of future hemostatic material technology which will help millions around the world. Initial in vitro experimentation focused on investigating the key chemical and structural material properties which affect Factor VII (FVII) activation in citrated human plasma. Enzyme-linked assays were utilized to confirm the ability of specifically formulated charged hydrogels to induce FVII activation and provided insight into the critical material parameters involved in this activation. Dynamic mechanical analysis was used to establish a correlation between polymeric microstructure and FVII activation. Experiments utilizing coagulation factor depleted and inhibited plasmas indicated that FVII, FX, FII, and FI are all vital to the process outlining the general mechanism of fibrin formation from the onset of FVII activation. The ability of the polymer to induce fibrin formation in "artificial plasma" explicitly lacking calcium, TF, and platelets suggested that a specifically designed material surface has the capability to substitute for these vital cofactors. Clinical diagnostic experimentation using sheep blood indicated that hydrogels containing higher amounts of electrostatic positive charge and lower cross-link density were able to induce faster, more robust clot formation in the presence of a coagulation cascade activator. Subsequent in vivo animal experimentation clearly demonstrated the ability of such hydrogels to aggregate platelets and erythrocytes promoting the formation of an effective hemostatic seal at the wound site. Moreover, in vivo testing confirmed the viability of such a charged polymer hydrogel to effectively control blood loss in a clinically relevant model

Modified U1snRNAs as innovative therapeutic strategy for inherited coagulation factor deficiencies

The theoretical framework in which this study is framed concerns the internationalization of production dynamics of firms abroad. The international literature in this field argues that there is not a conventional definition nor a universally tested theory that can explain all forms of foreign-owned production (see, among others, Dunning, 1988b and 1993a, Ietto-Gillies, 2005 and 2007). In this complex context, a large amount of Italian literature has been published around the theoretical framework of internationalization of production of Italian companies abroad (Tattara, Corò and Volpe, 2006, Mariotti and Mutinelli, 2005, et.al.). Nevertheless, a limited number of quantitative studies are available about the topic of Italian firms “migration” in Romania - as a particular expression of productive internationalization of our companies in this Country (Majocchi, 2002, Unimpresa Romania, 2005 and Antenna Veneto Romania, 2005). Thus, in order to investigate it by an industrial policy perspective a multilevel modelling approach has been applied. In particular, a two level model has been used to determine the effects of Romanian regions (romanian judets) in which Italian manufacturing firms internationalized the production in 2009 on their industrial performance. Based on a business register-based survey a business register of the Italian business community in Romania has been created. The data on Italian manufacturing firms located in Romania in 2009 have been selected from this register. The dataset used for the analysis includes 796 cases of firms internationalized in Romania. Therefore, a two level model has been carried out in order to determine the effects of intra-industry characteristics of these firms (i.e. Firm size and Firm Industrial specialization) on their industrial performance in 2009 both at individual and area level. The model results have shown that: as the firms dimension increase they have to be extremely sensible in the choice of the romanian region in which they want to (de)localize the production. Furthermore, the choice of the romanian region of productive delocalization has to be chosen by firms according to their sectors of activity

Design of small-molecule active-site inhibitors of the S1A family proteases as procoagulant and anticoagulant drugs

Vitamin K antagonists (VKA) have long been the default drugs for anticoagulant management in venous thrombosis. While efficacious, they are difficult to use due to interpatient dose–response variability and the risks of bleeding. The approval of fondaparinux, a heparin-derived factor Xa (fXa) inhibitor, provided validation for the development of direct oral anticoagulants (DOAC), and currently such inhibitors of thrombin and fXa are in clinical use. These agents can be used without regular coagulation monitoring, but the inherent risk of bleeding complications associated with blocking the common coagulation pathway remains. Efforts are now underway to develop DOACs that inhibit components of the intrinsic and extrinsic coagulation cascades upstream of thrombin and fX. Evidence from humans and from transgenic animal models suggests that this strategy may provide a better therapeutic margin between antithrombotic and antihemostatic effects. Here the design of active-site inhibitors of S1A proteases involved in coagulation and fibrinolysis is summarized

The carboxyl-terminal region of coagulation factors: role in biosynthesis and function of FVII and FX

-BACKGROUND- Factor VII (FVII), factor IX (FIX), factor X (FX) and protein C (PC), belonging to the family of coagulation vitamin K-dependent serine proteases, share high sequence and structural homology at the gene and protein level. However, their carboxyl-terminal region displays striking differences in length and aminoacid composition. While this region of FIX and PC has been demonstrated to be essential for efficient biosynthesis and secretion, little is shown for FVII and FX. -AIMS- The main aim was to investigate the carboxyl-terminal region of FVII and FX as determinant of biosynthesis/secretion and/or function. In the study we took advantage of the characterization of i) a natural variant of FVII characterized by a nonsense mutation (R402X) leading to a slightly truncated protein (-4 residues), and ii) natural anti-FVII inhibitory antibodies developed in a patient with an altered carboxyl-terminal region. The study was approached both by assays in patient’s plasma and by expression of the recombinant FVII variants in eukaryotic cells. To address the issue of the role of the carboxyl-terminal region of FX, a panel of progressively truncated FX variants has been expressed and characterized. -MAIN RESULTS AND CONCLUSIONS - The main results of the studies can be summarized as follows. i) Demonstration that the truncated FVII-402X molecule, albeit poorly secreted, possesses an increased specific activity, which explains the association of the R402X nonsense mutation with an asymptomatic phenotype; ii) identification of an anti-FVII inhibitory antibody in a patient homozygous for a frequent FVII frameshift mutation (11125delC) and data supporting the FVII carboxyl-terminal region as the main epitope of this antibody; iii) demonstration that the carboxyl-terminal region of FVII is essential for efficient biosynthesis and secretion, and the presence of an inverse relationship between the extent of the deletion of the carboxy-terminus and secretion levels. The deleted variants however possess a normal specific activity, thus not supporting a functional role for the very last residues of FVII; iv) demonstration that efficient secretion of FX, at variance from its highly homologous FVII, FIX and PC proteins, is not affected by short deletions (up to 21 residues) of the carboxyl-terminal region, which seems to have a functional role. Taken together these data indicate a differential role of the carboxyl-terminal region of FVII and FX, which might have contributed to divergence and evolution of these serine proteases from the common ancestor enzyme. -METHODOLOGICAL APPROACHES- Production and expression of recombinant proteins, ELISA-based and functional assays have been exploited in this study to investigate the expression and the activity of different natural or recombinant variants of coagulation FVII and FX

The Endothelial Protein C Receptor enhances FVIIa mediated hemostasis

Recombinant activated human Factor VII (rFVIIa) is an established hemostatic agent in hemophilia but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rFVIIa also interacts with the endothelial protein C receptor (EPCR) through its g-carboxyglutamic acid (Gla) domain with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rFVIIa hemostasis in hemophilia using a mouse model system. Murine activated FVII (mFVIIa) is functionally homologous to rFVIIa, but binds poorly to murine EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla-domain. The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. Using two macrovascular injury models in hemophilic mice, administered mFVIIa-FMR exhibited superior hemostatic properties compared to mFVIIa. These effects were specific to the mFVIIa- FMR and mEPCR interaction since antibody blocking of mEPCR abolished them. Since mFVIIa-FMR models both the TF-dependent as well as EPCR binding properties of rFVIIa, our data unmask a novel contribution of EPCR on the action of rFVIIa administration in hemophilia. This may prompt the rational design of improved and safer rFVIIa therapeutics

Engineering rFVIIa-loaded platelets; a novel approach to treating acute bleeding

Haemorrhage remains a leading cause of mortality around the world, resulting from both trauma and surgery. Current treatments for acute haemorrhage include blood products such as fresh frozen plasma, as well as platelet transfusion and recombinant clotting factors such as rFVIIa. However, the use of recombinant clotting factors is limited due to cost and adverse side effects resulting from increased thrombosis, such as increased rate of myocardial infarction and cerebrovascular accidents. Platelets are small anucleate cells that exist in very large quantities in our blood and, along with cross-linked fibrin from the coagulation cascade, are involved in forming a haemostatic plug upon exposure to damaged endothelium in a wound. They are metabolically active and undergo a process of platelet activation when stimulated by pro-thrombotic agonists such as thrombin resulting from the coagulation cascade. This then triggers a process whereby the contents of their granules are released locally to facilitate coagulation. This thesis explores the possibility of loading these platelet granules with recombinant clotting factors, in this case rFVIIa which has already been shown in clinical trials to result in a significant decrease in mortality from acute haemorrhage. The targeted delivery of this drug is explored, both through endocytosis and genetic engineering of megakaryocytes differentiated in vitro from induced pluripotent stem cells (iPSCs). These are evaluated with in vitro assays to model the process of clot formation, as well as an in vivo model of haemostasis to compare their efficacy to conventional treatments. Overall, this serves as a proof of concept of the engineering of platelet granules as a novel drug delivery system.Cambridge School of Clinical Medicine, the Rosetrees Trust and the Frank Edward Elmore Fun

Employing Template-Directed Assembly to Create a Novel Coagulation Assay

Blood coagulation is an important aspect of hemostasis in the human body. Under normal circumstances, the blood coagulates using two different pathways, the intrinsic and the extrinsic. The extrinsic pathway works to counteract trauma but may lead to stroke forming clots. Components for an assay were created so that an assay could be designed to test the functionality of the proteins involved in the clotting cascade: Tissue Factor, factor VII, and factor X when used in conjunction with Template Directed Assembly (TDA) on a nickel-nitriloacetic acid (Ni-NTA) derived liposome

Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo