Fibrin Network Porosity and Endo-/Exogenous Thrombin Cross-talk

The earliest assessment of fibrin network porosity used a liquid permeation system and confocal 3D microscopy, which was later replaced by scanning electron microscopy. Although the methods have extensively been applied in studies of health or disease, there remains debate on the choice of a proper clotting trigger. In this review, we assess published data and convey our opinions with regard to several issues. First, when the coagulation process is initiated by recombinant tissue factor (rTF) and phospholipids, the fibrin network porosity is regulated by the endogenous thrombin based on enzymatic activations of multiple coagulants. If purified thrombin (1.0 IU/mL) is employed as the clotting trigger, fibrin network porosity may be affected by exogenous thrombin, which directly polymerizes fibrinogen in plasma, and additionally by endogenous thrombin stemming from a “positive feedback loop” action of the added thrombin. Second, with use of either endogenous or exogenous thrombin, the concentration and clotting property of available fibrinogen both influence the fibrin network porosity. Third, in the assay systems in vitro, exogenous thrombin but not rTF-induced endogenous thrombin seems to be functional enough to activate factor XIII, which then contributes to a decrease in the fibrin network porosity. Fourth, fibrin network porosity determines the transport of fibrinolytic components into/through the clots and therefore serves as an indicator of the fibrinolysis potential in plasma.

Multiple thrombosis associated with Cytomegalovirus enterocolitis in an immunocompetent patient: a case report

Background Cytomegalovirus (CMV) is reported to have thrombogenic characteristics that activate factor X in vitro and stimulate the production of factor VIII and von Willebrand factor (vWF). Thrombosis associated with CMV infection is prevalent among immunocompromised patients and predominantly presents as a solitary large thrombus in the deep vein, pulmonary artery, splanchnic arteriovenous ducts, or other similar sites. Multiple thrombi, however, are rarely observed in such cases. Here, we report about an immunocompetent man with multiple microthrombi associated with CMV infection. Case presentation A 72-year-old Japanese man who complained of abdominal pain was hospitalized with multiple colonic stenosis. He was later diagnosed with CMV enterocolitis and treated with ganciclover from Day 27 post-admission. During hospitalization, the patient developed thrombi in his fingers. He was initially treated with anticoagulant therapy (rivaroxaban); however, the therapy was discontinued owing to a prolonged activated thromboplastin time and an elevated international normalized ratio of prothrombin time. Instead, vitamin K and fresh-frozen plasma were administered. Nevertheless, his coagulation profile remained abnormal. Eventually, he developed colonic perforation and had to undergo emergency surgery. An intraoperative specimen showed several microthrombi in the middle and small arteriovenous ducts of his small and large intestines. The patient’s coagulopathy improved preoperatively, and his overall condition improved postoperatively. Since the activation of ADAMTS13 was reduced remarkably, the thrombotic tendency was determined to be a thrombotic microangiopathy-like condition owing to increased vWF. We could not attribute the coagulopathy to any other cause except CMV infection; therefore, we concluded that this was a case of multiple thrombosis associated with CMV. Conclusions We present an extremely rare case of a patient with multiple thrombotic microangiopathy-like microthrombosis caused by CMV infection. Our findings suggest that CMV infection may be considered as a differential diagnosis for immunocompetent individuals who present with thrombosis of unspecified cause.

Montelukast - Its Immunomodulatory and Antiviral Action in COVID-19

Coronavirus disease-19 (COVID-19) is the deadliest pandemic that the whole world is facing today. COVID-19 is different from normal flu by its two lethal manifestations which includes deadly pneumonia which may lead to acute respiratory distress syndrome (ARDS) due to hyper-inflammation of alveolar tissues and pulmonary intravascular coagulopathy (PIC).1,2 It is noteworthy here to mention that both these lethal manifestations of COVID-19 are due to abnormally high levels of pro-inflammatory cytokines like interleukin (IL) - 1β, IL - 6, and tumour necrosis factor (TNF) - α, termed as “cytokine storm.”3,4 There is a certain link between pro-inflammatory cytokines like IL - 1β, IL - 6, and TNF - α and its pro-coagulatory influence on coagulation pathway mediated by tissue factor that binds and activate factor VII, leading to formation of tissue factor – VII a complexes which results in the activation of clotting factor X and IX.4 Recently the researchers in China and some European countries have found raised level of pro-inflammatory cytokines particularly IL - 6 in severe cases of COVID-19. They also found raised D-dimer, fibrinogen levels and prothrombin time in moderate to severe COVID-19 cases.5,6 Both of these lethal manifestations of COVID-19 – ARDS and PIC are linked to raised levels of pro-inflammatory cytokines, particularly, IL - 6. It is not very clear that the pro-inflammatory action of cytokines is mediated through leukotrienes as the biochemical assay for leukotrienes are not widely available but possibility of this probable mechanism cannot be ruled out. Hence, development of any molecule with ability to inhibit pro-inflammatory cytokines, particularly IL-6 may be able to tame the lethal nature of COVID-19, and may ultimately reduce the mortality of this deadly pandemic. Montelukast sodium is such molecule which has capacity to inhibit proinflammatory cytokines such as IL - 1β, IL - 6, and TNF - α.7 Montelukast sodium is leukotriene receptor antagonist that inhibits the cysteinyl leukotriene type-1 receptor. Leukotrienes modulate the production of pro-inflammatory cytokines.8 Its antagonist action on leukotriene receptors can inhibit the production of these pro-inflammatory cytokines. Even recent in silico study by Jacobson at Oak Ridge National Lab, was found that excess bradykinin production may be responsible for pulmonary, cardiac, neurological and nephrological lethal manifestations of COVID-19.9 Crimi et al.10 already found that Montelukast is effective to control bradykinin induced bronchoconstriction. Thus, theoretically, montelukast seems to be best molecule to deal with deadly manifestation of COVID-19 even if we go by cytokine storm hypothesis or bradykinin hypothesis.

Effect of External Activate Factors Serving as Clue

Why is there a demand for consultants and outside directors? That is because what they are asked for objective advice and the clue—this chapter describes them as external activate factors—is precious. This chapter focuses on how the external activate factor affects human creativity, especially the process of creating new products or services, and the author conducted an experiment to compare the effect of external activate factors at a lecture of graduate school. As a result, when the external activate factor is provided, the emergence of abduction and new rules was observed, and the effect of storytelling was confirmed. Further research is to find what kind of external activate factor is preferable.

Identification of novel glycosylation events on human serum-derived Factor IX

ABSTRACTHuman Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.

Coagulation properties of erythrocyte derived membrane microparticles

Membrane microparticles (MP) produced upon cell activation and/or damage possess coagulation activity, i.e. ability to accelerate blood clotting. They contain on their surface phosphatidylserine (PS), a substrate for assembling coagulation enzymatic complexes, and some of them tissue factor (TF), the initiator of clotting cascade reactions. In this study coagulation properties of MP derived from erythrocytes have been investigated. These MP were obtained from donor's erythrocytes activated with ionophore A23187 as well as from outdated erythrocyte concentrates for transfusion. MP were counted by flow cytometry. Coagulation activity of MP was examined by modified plasma recalcification assay. Involvement of PS and TF in this reaction was assessed using PS blocker lactadherin and anti-TF antibodies. TF activity in MP was measured by its ability to activate factor X in a chromogenic assay. Size of MP was evaluated by dynamic light scattering. Properties of erythrocyte MP were compared with previously characterized (using the same methodological approaches) MP derived from platelets and monocytic THP-1 cells, lacking and containing TF, respectively. Erythrocyte MP accelerated plasma clotting, but less actively than MP from platelets and MP from THP-1 cells, which demonstrated maximal activity. Lactadherin completely inhibited coagulation activity of all MP. Anti-TF antibodies did not affect clotting parameters in the presence of platelet and erythrocyte MP, but slowed clotting in the presence of MP from THP-1 cells. TF activity was not detected in erythrocyte and platelet MP, unlike MP from THP-1 cells expressing active TF. MP derived from erythrocytes were smaller than MP from platelets and THP-1 cells, with average diameter about 200 nm and 400 nm respectively. Thus, MP from erythrocyte possess less ability to accelerate plasma clotting in comparison with MP from platelet and THP-1 cells. The data obtained suggest that lesser coagulation activity of erythrocyte MP in comparison with MP from THP-1 cells is due to the absence of TF in erythrocyte MP (in contrast to MP from THP-1 cells) and to their smaller size, and in comparison with MP from platelets (which as erythrocyte MP do not express TF) is due to their smaller size only.

A Novel Variant of Factor VIII Yields Cofactor Activity without Proteolysis between the A1 and A2 Domains

Factor VIII (FVIII) has a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2), and intracellular processing within the B domain results in its secretion as a heterodimeric procofactor consisting of a variably sized heavy chain (A1-a1-A2-a2-B) and a light chain (a3-A3-C1-C2). Proteolytic cleavage by thrombin at R372, R740, and R1689 removes the B-domain, releases it from vWF by cleaving the a3-acidic region and activates FVIII to the heterotrimeric cofactor, FVIIIa (A1-a1/A2-a2/A3-C1-C2). The requirement for cleavage following a1 to generate active cofactor sets FVIII/FVIIIa apart from factor V, its structural and functional homolog. FVIII binds its cognate protease, factor IXa (FIXa) with high affinity in a membrane-dependent way. However, this complex does not efficiently activate factor X (FX) until R372 is cleaved, indicating that this cleavage reaction is important in facilitating FX recognition and its enhanced activation by intrinsic tenase complex. We speculated that separation of A1-a1 and A2-a2 domains through cleavage at R372 might be replicated by inserting a linker between A1-a1 and A2-a2 domains even when the 372 site was rendered uncleavable. We chose canine FVIII (cFVIII) to test our ideas due to its higher expression levels in cell culture compared to human FVIII. We made cDNA constructs encoding cFVIII variants with flexible (GGGGS, [GGGGS]3, GGGGGG) or rigid (EAAAK, [EAAAK]3, PAPAP) linkers between A1-a1 and A2-a2 domains on the R366Q backbone (corresponding to R372 in human FVIII). All constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Clotting times of cFVIII variants with flexible linkers were very similar to cFVIII R366Q without linker (~64 sec). In contrast, clotting times of cFVIII variants with rigid linkers were consistently lower (16 - 26 sec). cFVIII variants (R366Q, R366Q-EAAAK, R366Q-PAPAP) were purified from 15L of expression media each. In one stage aPTT assays purified cFVIII variants (10 nM) showed clotting times (R366Q-EAAAK, 33.3 s, R366Q-PAPAP, 26 s) that were considerably shorter than the R366Q variant (52.2 s) and more in line with wild type cFVIII (cFVIII-WT,19.3 s). To assess possible cleavage at the 366 site, purified cFVIII variants were analyzed by SDS-PAGE following treatment with a high concentration of thrombin. As expected, cFVIII-WT was quantitatively cleaved at R366, R734 and R1689 to produce bands corresponding to A1-a1, A2-a2 and A3-C1-C2. In contrast, both R366Q and R366Q-PAPAP variants were resistant to cleavage at 366, yielding bands corresponding to A1-a1-A2-a2 and A3-C1-C2. The cFVIII-R366Q-EAAAK variant was susceptible to some cleavage, likely at the lysine present within linker. We also performed steady state kinetic studies of FX activation using limiting concentrations of IXa, saturating concentrations of cFVIII variants pretreated with thrombin, membranes and increasing concentrations of FX. Vmax/[E] for FXa formation was 140 ± 7 min-1 with cFVIIIa-WT, 4 ± 0.3 min-1 with cFVIIIa-R366Q, and 42 ± 4 min-1 with cFVIIIa-R366Q-PAPAP. The Km for FX remained unaffected for all variants. The results indicate that the impaired cofactor activity of the FVIII variant that is not cleaved at the 366 site can be substantially rescued by insertion of the PAPAP linker after 366Q. Further optimization of the linker might completely correct defective FX activation associated with the lack of proteolytic cleavage between A1-a1 and A2-a2 domains. Our study indicates that cleavage at the 366 site is not an absolute determinant of FVIII activation and provides new insights into the function of FVIIIa as a cofactor within the intrinsic tenase complex. Disclosures Goolyam Basavaraj: Bayer: Research Funding; Grifols: Research Funding. Krishnaswamy:Portola: Research Funding; Baxalta: Consultancy; Janssen Research & Development: Research Funding.

Anticoagulant and Antithrombotic Properties of Three Structurally Correlated Sea Urchin Sulfated Glycans and Their Low-Molecular-Weight Derivatives

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.

Characterization of IXINITY® (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph

The goal of these studies was to extensively characterize the first recombinant FIX therapeutic corresponding to the threonine-148 (Thr-148) polymorph, IXINITY (trenonacog alfa [coagulation factor IX (recombinant)]). Gel electrophoresis, circular dichroism, and gel filtration were used to determine purity and confirm structure. Chromatographic and mass spectrometry techniques were used to identify and quantify posttranslational modifications. Activity was assessed as the ability to activate factor X (FX) both with and without factor VIIIa (FVIIIa) and in a standard clotting assay. All results were consistent across multiple lots. Trenonacog alfa migrated as a single band on Coomassie-stained gels; activity assays were normal and showed <0.002 IU of activated factor IX (FIXa) per IU of FIX. The molecule has >97% γ-carboxylation and underwent the appropriate structural change upon binding calcium ions. Trenonacog alfa was activated normally with factor XIa (FXIa); once activated it bound to FVIIIa and FXa. When activated to FIXa, it was inhibited efficiently by antithrombin. Glycosylation patterns were similar to plasma-derived FIX with sialic acid content consistent with the literature reports of good pharmacokinetic performance. These studies have shown that trenonacog alfa is a highly pure product with a primary sequence and posttranslational modifications consistent with the common Thr-148 polymorphism of plasma-derived FIX.