ENDOSCOPIC TREATMENT OF ESOPHAGEAL-BRONCHIAL FISTULA IN A PATIENT WITH A LEYDEN MUTATION

Bronchoesophageal Fistula (BEF) is an uncommon condition related to complex thoracic surgery. The development of BEF is usually an indication of the progression of bronchial or lung cancer; whereas the etiology of this illness being recorded as benign is much rarer – not exceeding 4 - 6%. [1, 2]. Surgery is the main method for treating patients who have benign BEF, allowing for the reliable result. At the same time, similar operations are extremely traumatic and are fraught with the development of post-operation complications, especially when concerning patients with genetically determined coagulopathy. Factor V Leiden mutation is a hereditary coagulopathy in which there is a point mutation in the gene that encodes blood coagulation factor V. Factor V Leiden mutation is the most common cause of the hereditary disposition to thrombosis, heart attacks, strokes in Europeans. The frequency of occurence among the population of the USA is 4 – 6% [3]. We present our own observation of successful endoscopic surgery to treat BEF in a patient with Factor V Leiden mutation and chronic pulmonary embolism.

Diagnosis and choice of haemostatic therapy during surgery in patients with combined coagulation factor V and VIII defi ciency

Introduction. Among the most common congenital coagulopathies are haemophilia and Von Willebrand disease. These illnesses are often mimicked by orphan hereditary coagulopathies, including combined coagulation factor V and VIII deficiency.Aim — description of a clinical presentation, hampered diagnosis and choice of haemostatic therapy in a surgical patient with combined blood coagulation factor V and VIII deficiency.Main findings. We describe a clinical case of congenital combined factor V and VIII deficiency and detail the aetiology, frequency, localisation and intensity of haemorrhages. Comorbidity and surgical indications are demonstrated to require an inter-specialty medical involvement.

The Procoagulant Snake Venom Serine Protease Potentially Having a Dual, Blood Coagulation Factor V and X-Activating Activity

A procoagulant snake venom serine protease was isolated from the venom of the nose-horned viper (Vipera ammodytes ammodytes). This 34 kDa glycoprotein, termed VaaSP-VX, possesses five kDa N-linked carbohydrates. Amino acid sequencing showed VaaSP-VX to be a chymotrypsin-like serine protease. Structurally, it is highly homologous to VaaSP-6 from the same venom and to nikobin from the venom of Vipera nikolskii, neither of which have known functions. VaaSP-VX does not affect platelets. The specific proteolysis of blood coagulation factors X and V by VaaSP-VX suggests that its blood-coagulation-inducing effect is due to its ability to activate these two blood coagulation factors, which following activation, combine to form the prothrombinase complex. VaaSP-VX may thus represent the first example of a serine protease with such a dual activity, which makes it a highly suitable candidate to replace diluted Russell’s viper venom in lupus anticoagulant testing, thus achieving greater reliability of the analysis. As a blood-coagulation-promoting substance that is resistant to serpin inhibition, VaaSP-VX is also interesting from the therapeutic point of view for treating patients suffering from hemophilia.

New Advances Along the Common Coagulation Pathway

Hemostasis is achieved through spatially and temporally regulated thrombin generation following vascular injury. Blood coagulation factor V plays an important role in this process as it has a major impact on thrombin production. FV is a large, heavily glycosylated protein sharing homology and domain organization with FVIII (A1-A2-B-A3-C1-C2). It is an inactive procofactor and must be cleaved by thrombin or other proteases to remove the B-domain to yield activated FV (FVa). FVa is a cofactor for FXa in the prothrombinase complex, the enzyme that activates prothrombin. Given the profound effect FVa has on thrombin formation, FV activation represents an important step in hemostasis. Mechanistic studies have revealed that proteolysis within the FV B-domain serves to remove key autoinhibitory sequences that serve to conceal/block FXa binding site(s). These sequences consist of an evolutionary conserved basic region (BR) in the middle of the B-domain and flanking acidic regions (AR1 and AR2) which define the minimal sequences necessary to maintain FV as a procofactor. Removal of either of these regions results in a molecule that can function in prothrombinase. These sequences are intact and present in plasma FV; however, FV released by activated platelets and FV cleaved by FXa represent active forms FV(a) that lack the BR. Recent work indicates these forms of FV(a) may be regulated by TFPIathrough molecular mimicry. TFPIahas a C-terminal basic tail which shares remarkable homology to the FV BR suggesting TFPIamay be an important regulator at the level of prothrombinase. Remarkably, another form of FV missing the BR, resulting from alternative splicing, has been recently described. The FV-East Texas, FV-Amsterdam, and FV-Atlanta alterations all result in the enhanced production of an alternatively spliced form of FV (FV-short) that has most of the B-domain removed including the BR. While these forms of FV should be constitutively active, studies have shown that FV-short circulates in complex with TFPIa. Further, in each of the clinical cases, TFPIalevels are substantially elevated resulting in a bleeding phenotype. While there are several remaining unanswered questions, the FV(a)-TFPIα interaction as well as the identification of FV-short, has the potential to fundamentally alter our understanding of the regulation of the initiation of coagulation. Disclosures Camire: Bayer: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Conserved Structural Motifs Cooperate to Maintain Blood Coagulation Factor V in An Inactive Procofactor State.

Abstract 850 Blood coagulation factor V (FV) is a multi-domain protein which circulates as an inactive procofactor and has high structural homology with factor VIII. To express procoagulant activity, FV must be proteolytically processed within its central B-domain (836 residues) with thrombin being considered the key physiological activator. Following liberation of the B-domain (residues 710-1545), activated FV (FVa) functions as a cofactor for factor Xa within the prothrombinase complex and dramatically enhances the rate of thrombin generation. The central role which FVa assumes in prothrombinase indicates that its activation must be a key regulatory step in hemostasis. Although the proteolytic events that lead to the activation of FV have been well studied, the molecular mechanism by which B-domain release facilitates the procofactor to cofactor transition is not well understood. Recently, we have shown that in the absence of intentional proteolysis, deletion or substitution of discrete B-domain sequences drives the expression of procoagulant function (JBC, 282, 15030-9, 2007). Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids (963-1008) which is highly basic and well conserved, even though most of the B-domain has weak homology within the vertebrate lineage. In the current study, we examined if this basic B-domain region is sufficient to preserve FV as an inactive procofactor. To investigate this, the basic region (46 residues) was incorporated within the short B-domain of a previously characterized FV variant, FV-810. Factor V-810 has amino acids 811-1491 within the B-domain deleted and is a constitutively active cofactor, with functional properties equivalent to FVa. Using a PT-based clotting assay, purified prothrombinase assay, and direct fluorescent binding measurements with FXa-membranes we found that insertion of the basic region into FV-810 (inserted after residue 810) converted this cofactor-like species back to the procofactor-like state, despite >75% of the B-domain being absent. Next, using this new variant (FV+BR; B-domain of 201 residues), we assessed whether residual B-domain sequences within FV+BR contribute to maintaining FV in an inactive, procofactor state. Elimination of ∼100 residues on the N-terminal side of FV+BR was without functional consequence; that is, the procofactor state was maintained. In contrast, removal of B-domain sequences (∼50 residues, 30% of which are acidic) to the C-terminal side of the basic region shifted FV-810+BR from an inactive procofactor to an active cofactor. As expected, all purified FV derivatives exhibited full cofactor activity following treatment with thrombin. Together, these data show that B-domain sequences 963-1008 (basic region) appear to work in concert with the acidic C-terminal region of the B-domain (1492-1545) to keep FV in an inactive procofator state. These sequence elements appear to be necessary and sufficient as we were able to construct a FV variant with a B-domain length of only 103 amino acids that remarkably still had procofactor-like properties. Interestingly, these two regions of the B-domain (963-1008 and 1492-1545) are generally well conserved throughout the vertebrate lineage, while the remaining regions of the B-domain are not. We speculate that these B-domain sequences bind intramolecularly to heavy and/or light chain sequences thereby concealing critical binding sites on the FV molecule which govern the function of the active cofactor species. Disclosures: Camire: Wyeth: Patents & Royalties, Research Funding.

An Assay to Quantify the Two Plasma Isoforms of Factor V

SummaryBlood coagulation factor V (FV) circulates in the blood in two forms, designated FV1 and FV2. In model systems containing purified proteins FV1 appears to be more thrombogenic than FV2. Recently, we reported that in plasma from carriers of the R2 haplotype, a polymorphism which encodes several amino acid changes in FV and which is associated with an increased risk of thrombosis, the FV1/FV2 ratio is shifted in favor of the more thrombogenic form FV1. Here we describe in detail the assay that enables quantification of the plasma levels of FV1 and FV2. FV present in highly diluted plasma samples was activated with thrombin and the FVa generated was subsequently quantified in two prothrombinase-based assay systems. In the first assay, which is performed at saturating amounts of FXa and phospholipid vesicles with a high mole fraction phosphatidylserine, FVa1 and FVa2 express the same cofactor activity in prothrombin activation. Hence, this assay quantifies the total FV level (FV1 + FV2) present in plasma. In the second assay, which is performed at suboptimal amounts of FXa and phospholipid vesicles with a low mole fraction phosphatidylserine, FVa2 has approximately an 8-fold higher cofactor activity than FVa1. Therefore, the response in this assay depends on the relative amounts of FV1 and FV2 in the plasma sample. Calibration curves made with samples containing known concentrations of purified FVa1 and FVa2 subsequently allowed calculation of the amounts of FV1 and FV2 present in plasma.