Conversion Of Fibrinogen Research Articles

Protein composition of clots detected in pooled cryoprecipitate units

On rare occasions, upon thawing of stored cryoprecipitate components, clots are observed on visual inspection. Although it has been assumed that the clot reflects fibrinogen to fibrin conversion, there are few published studies that document that this assumption is correct. Our studies were conducted to further identify the protein characteristics of the clotted material. Clotted material isolated from four thawed cryoprecipitate pools was examined by solubilization procedures and electrophoresis analysis. Solubilization of much of the clotted material in phosphate-buffered saline warmed to 37°C suggested the presence of soluble fibrin. Gel electrophoresis under reducing conditions showed that the most prevalent bands exhibited molecular weights corresponding to the α, β, and γ subunits of fibrinogen with a much lighter band exhibiting the molecular weight of fibrinogen γ-γ dimer, consistent with the presence of partially crosslinked fibrin. The presence of the dimer indicated that the clotted material was caused by the action of thrombin, but also reflected the action of Factor XIIIa. No ongoing clot formation was observed. Our studies indicate that, on rare occasions, fibrinogen conversion to fibrin is responsible for observable clots in thawed cryoprecipitate pools. These clots are structurally heterogeneous, including both noncrosslinked (soluble) and crosslinked (insoluble) fibrin. This diversity in the fibrin structure may account for some of the diversity in the limited literature regarding their presence in cryoprecipitate pools.

Modeling of human factor Va inactivation by activated protein C

BackgroundBecause understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (F)Xa and its cofactor FVa) plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC), an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and “protection” of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation.ResultsA model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative analysis of in vitro experiments and mathematical constructs we are able to produce a final validated model that includes 24 chemical reactions and interactions with 14 unique rate constants which describe the flux in concentrations of 24 species.ConclusionThis study highlights the complexity of the inactivation process and provides a module of equations describing the Protein C pathway that can be integrated into existing comprehensive mathematical models describing tissue factor initiated coagulation.

Spectroscopic and Electrochemical Detection of Thrombin/5'-SH or 3'-SH Aptamer Immobilized on (porous) Gold Substrates

Thrombin is a serine protease that catalyzes the conversion of soluble fibrinogen to insoluble fibrin, and thus induces physiological and pathological blood coagulation. Therefore, it is important to detect thrombin in blood serum for purposes of diagnosis. To achieve this goal, it has been suggested that a 15-mer aptamer strongly binds with thrombin to form a G-quartet structure of the aptamer. Generally, 5'-end thiol-functionalized aptamer has been used as an anti-thrombin binder. Herein, we evaluate the possibility of utilizing a 3'-SH aptasensor for thrombin detection using SPR spectroscopy, and compare the enhancement of the electrochemical signal of the thrombin-aptamer bound on a porous gold substrate. Although the two aptamers have similar configurations, in SPR analysis, the 3'-SH aptamer was a effective aptasensor as well as 5'-SH aptamer. Results from electrochemical analysis showed that the porous gold substrate acted as a good substrate for an aptasensor and demonstrated 5-fold enhancement of current change, as compared to gold thin film.

Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation

Interactions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus.

Plasminogen and fibrinogen plasma levels in coronary artery disease

The process of haemostasis ensures that following vascular damage blood remains confined to the circulatory system. This is not a single biological pathway, but rather a complex interplay of several distinct processes(1). Injury to the endothelium causes the transition of endothelial surface from a non-thrombogenic state to one actively promoting thrombus formation. The process starts when injury causes a breach in the endothelium with exposure of subendothelial collagen and tissue factor. Collagen activates platelets and tissue factor exposure initiates the coagulation cascade - both processes lead to thrombus formationthat is localised at the site of injury. While the end product is the generation of fibrin from fibrinogen, the composition of thrombi in the arterial and venous circulations is dissimilar. In the arterial system the thrombus predominantly comprises of platelets and fibrin whereas in the venous system fibrin together with red cells and white cells are the major components of the clot. Regardless of the site of clot, the formation of a stable fibrin network is the ultimate step in thrombus formation. The conversion of fibrinogen to fibrin is brought about by cleavage of fibrinogen. This process is undertaken by thrombin which cleaves fibrinogen into two smaller peptides, fibrinopeptide A and B. Fibrin monomers are then formed with subsequent polymerization. Finally, the clot is consolidated when factor XIII cross-links the fibrin monomers. Once the clot has formed and the fibrinolytic system begins the proteolytic removal of thrombi to ensure that vessel patency is restored. Plasminogen binds to fibrin and is activated by tissue plasminogen activator (tPA) to plasmin. Plasmin begins the dissolution of fibrin although; because of its broad specificity it causes degradation of fibrinogen as well. Just like the coagulation cascade, the fibrinolytic system is a complex interplay of activators and inhibitors to ensure optimal haemostasis. Obviously, in disease the fine balance of haemostasis is disturbed. Atherosclerosis is a multifactorial disease where vascular injury leads to lipid accumulation and deposition of platelets and fibrin. It is not surprising therefore, that disorders of different components of haemostasis can contribute to the pathogenesis of atherosclerosis. The current study compared the level of fibrinogen and plasminogen in angiographically normal subjects and those with mild/moderate, and severe coronary artery disease(2). A graded correlation was found between levels of fibrinogen and the severity of coronary artery disease. Elevated levels of plasma fibrinogen have been shown to be an independent risk factor for coronary artery disease(3,4). Whether this is a direct effect of hyperfibrinogenemia is unclear, although some evidence exists to suggest that elevated fibrinogen may affect the process of atherogenesis by fibrin deposition within developing atherosclerotic lesions(5). Because fibrinogen has also been associated with other traditional cardiovascular risk factors, an atherothrombotic effect mediated through a raised fibrinogen level would appear plausible. This would be indirectly supported by thrombotic association of dysfibrinogenemia(6), a condition which primarily produces a bleeding tendency but is well known to cause thrombotic events too. There are several mechanisms by which fibrinogen may increase cardiovascular risk. It binds specifically to activated platelets via glycoprotein IIb/IIIa, contributing to platelet aggregation. Elevated fibrinogen levels increase plasma viscosity and promote fibrin formation. Chronic inflammatory conditions are known to result in accelerated atherosclerosis. That fibrinogen is an acute-phase reactant with levels rising significantly in inflammatory states may be pathogenetically relevant. Like disorders of fibrinogen impaired fibrinolysis appears to have a role in thrombosis. Plasmin, which results from the cleavage of plasminogen by plasminogen activators (PAs), predominantly mediates the intravascular clearance of fibrin. The important role of plasminogen in fibrinolysis makes it an interesting parameter to evaluate in various diseases. It is recognized that a decreased plasminogen level may affect the fibrinolytic activity and result in thrombotic tendency(7). It is therefore, not surprising that plasminogen levels were not found to be significantly elevated in patients with coronary artery disease. It is well known that increased levels of plasminogen activator inhibitor-1 (PAI-1) are found in myocardial infarction(8). A raised PAI-1 level would inhibit fibrinolysis by inhibiting tissue plasminogen activator and this may result in thrombotic tendency. It would have been interesting to look at PAI-1 levels as well and this aspect needs to be considered in future studies. While this paper reaffirms the association of elevated fibrinogen with coronary artery disease, in view of the multifaceted nature of atherosclerosis, especially the emerging role of inflammation in its pathogenesis, information on inflammatory markers would have been valuable(9,10). Further follow-up on the three cohorts with serial measurements of different parameters would give additional insight into correlating the progression of the disease with different haemostatic and fibrinolytic parameters.

Fibrinogen replacement therapy: a critical review of the literature.

Fibrinogen is a plasma glycoprotein with a molecular weight of 340 kDa; it is synthesised by the liver. The conversion of fibrinogen to fibrin is catalysed by thrombin and plays a key role in clot formation and stabilisation. In addition, fibrinogen induces platelet activation and aggregation by binding to the platelet fibrinogen receptor glycoprotein GPIIb/IIIa1. Fibrinogen replacement therapy is currently indicated as prophylaxis and therapy of haemorrhage in congenital and acquired fibrinogen deficiency, this latter being associated with liver failure, disseminated intravascular coagulation, massive transfusion and cardiac surgery)2,3. Hypofibrinogenaemia is defined by a decreased level of normal fibrinogen between 0.5 g/L and the lower limit of the normal range for the local laboratory (usually 1.5 g/L)4. Fibrinogen supplementation can be provided by transfusion of fresh-frozen plasma (FFP), cryoprecipitate and fibrinogen concentrate5,6. However, as FFP has several limitations including a low fibrinogen content, which means that large volumes must be given, and the risk of transfusion-related complications (e.g., transfusion-related acute lung injury [TRALI] and viral transmission), this critical analysis is focused on the role of cryoprecipitate and fibrinogen concentrate.

Dietary Spinach Saponin-Enriched Lipophilic Fraction Inhibits Platelet Aggregation and Blood Coagulation

In this study, we investigated the effect of spinach saponin-enriched lipophilic fraction (SSEF) on collagen (10 μg/mL)-stimulated platelet aggregation in vivo. Dietary SSEF dose-dependently inhibited collagen-induced platelet aggregation by decreasing thromboxane A₂ production and intracellular Ca²⁺ agonist activity as an aggregation-inducing autacoidal molecule. In addition, SSEF significantly increased the formation of cyclic AMP and cyclic GMP, intracellular Ca²⁺ antagonists that are aggregation-inhibiting molecules in collagen-stimulated platelets. These results suggest that SSEF is a potent inhibitor of collagen-stimulated platelet aggregation in vivo. Prothrombin time and activated partial thromboplastin time, indicators of blood coagulation, were potently prolonged by dietary SSEF in vivo. These findings suggest that SSEF prolongs the interval time between the conversion of fibrinogen to fibrin. Dietary SSEF also inhibited 0.4 M sucrose-induced hemolysis. Accordingly, our data demonstrate that SSEF might be a useful tool for inhibiting platelet activation and blood coagulation in thrombotic diseases.

Molecular Mechanisms Affecting Fibrin Structure and Stability

Fibrin structure and stability have been linked to many thrombotic diseases, including venous thromboembolism. Analysis of the molecular mechanisms that affect fibrin structure and stability became possible when the crystal structure of fibrinogen was solved. Biochemical studies of natural and recombinant variant fibrinogens have examined the interactions that mediate the conversion of soluble fibrinogen to the insoluble fibrin network. These studies identified intermolecular interactions that control fibrin structure, although some critical events remain ambiguous. Studies show that fibrin structure modulates the enzymatic lysis of the fibrin network, so the molecular mechanisms that control structure also control stability. Studies show that the mechanical stability of the fibrin clot depends on the properties of the fibrin monomer, leading investigators to explore the molecular basis of the monomer's mechanical properties. The work summarized here provides insights that might allow the development of pharmaceuticals and treatments to modulate fibrin structure and stability in vivo and thereby prevent or limit thrombotic disease.

New drugs: Dabigatran etexilate mesylate, fingolimod hydrochloride, and ulipristal acetate

Anticoagulant Atrial fibrillation is one of the most common types of abnormal heart rhythm, affecting more than 2 million Americans. Patients with atrial fibrillation are at greater risk of developing blood clots and at an estimated fivefold increased risk of experiencing a stroke. For many years, the vitamin K antagonist warfarin (e.g., Coumadin) has been the standard treatment for preventing these potentially disabling, and even fatal, complications, but its use is associated with serious adverse events and drug interactions and requires very close monitoring. This situation has provided the impetus for the development of new orally administered anticoagulants that provide benefits via a different mechanism of action. The most promising of these investigational agents are the ones that act as direct thrombin inhibitors or factor Xa inhibitors. Thrombin (serine protease) is an enzyme that enables the conversion of fibrinogen to fibrin during the coagulation cascade, a process that may result in the development of a thrombus. Dabigatran etexilate mesylate (Pradaxa— Boehringer Ingelheim), a direct thrombin inhibitor, is the first of the group of investigational oral anticoagulants to be approved in the United States. It is absorbed as the dabigatran etexilate ester that is then hydrolyzed to dabigatran, the active moiety. Dabigatran is metabolized to four different acyl glucuronides that have pharmacological activity similar to that of the parent compound. The drug and its acyl glucuronides inhibit both free and clot-bound thrombin and inhibit thrombin-induced platelet aggregation. Dabigatran is indicated specifically for reducing the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. Its effectiveness and safety were evaluated in a clinical trial that included more than 18,000 patients, in which patients received warfarin, dabigatran 150 mg twice a day, or dabigatran 110 mg twice a day. When used in the dosage of 150 mg twice a day, dabigatran reduced stroke and systemic embolism by 35% beyond the reduction attained with warfarin. The risk of major bleeding events generally was similar in the two groups. The 110-mg-twice-a-day regimen of dabigatran was determined to be noninferior to warfarin and less likely to cause bleeding events. However, this regimen is not identified in the dosage recommendations in the labeling, and the available capsule potencies (75 and 150 mg) do not facilitate the use of doses of 110 mg. The concern underlying the Food and Drug Administration decision to not approve, at least initially, a product containing 110 mg is that some prescribers might be overly cautious and not prescribe the 150-mg-twice-a-day regimen, which provides the greatest benefit in reducing the risk of stroke. The labeled indication for dabigatran is more limited than indications for warfarin, which also is approved for the prophylaxis and/or treatment of thromboembolic complications associated with cardiac valve replacement, the reduction of the risk of death, recurrent myocardial infarction (MI), thromboembolic events after an MI, prophylaxis and/or treatment of venous thrombosis and its extension, and pulmonary embolism. Dabigatran has been approved in some other countries for the prevention of venous thromboembolism in patients who have had joint replacement surgery. However, this is not a labeled indication in the United States currently. The most important concern with the use of dabigatran is the risk of bleeding, and the new drug is contraindicated in patients with active pathological bleeding. The risk of major bleeding events generally was similar with dabigatran (150 mg twice a day) and warfarin, with the exception of patients 75 years or older, in whom a higher incidence of bleeding with dabigatran occurred. A higher rate of major gastrointestinal (GI) bleeding events also was observed in patients treated with dabigatran. The risk of bleeding is increased by the concurrent use of other medications such as heparin, antiplatelet agents, and the chronic use of nonsteroidal anti-inflammatory drugs. Because of the increased risk of bleeding, patients who are to have surgical or invasive procedures performed should discontinue dabigatran at least 1 to 2 days before the procedure, if possible. The bleeding risk can be assessed by the ecarin clotting time (ECT). ECT is a better marker of the anticoagulant activity of dabigatran than activated partial thromboplastin time (aPTT), prothrombin time (PT)/international normalized ratio (INR), or thrombin time (TT). If ECT is not available, the aPTT test provides an approximation of the drug’s anticoagulant activity. At recommended therapeutic doses, dabigatran prolongs the aPTT, ECT, and TT. The INR test is relatively insensitive to the activity of the new drug and may or may not be elevated by its use. Approximately 35% of patients treated with dabigatran (150 mg twice a day) experienced GI adverse events compared with 24% of those treated with

The fibrinogen gamma 10034C > T polymorphism is not associated with Peripheral Arterial Disease

Conversion of fibrinogen to fibrin plays an essential role in hemostasis and results in stabilization of the fibrin clot. Fibrinogen consists of three pairs of non-identical polypeptide chains, encoded by different genes (fibrinogen alpha [FGA], fibrinogen beta [FGB] and fibrinogen gamma [FGG]). A functional single nucleotide polymorphism (SNP) in the 3' untranslated region of the FGG gene (FGG 10034C > T, rs2066865) has been associated with deep venous thrombosis and myocardial infarction. Aim of the present study was to analyze the role of this polymorphism in peripheral arterial disease (PAD). The study was designed as case-control study including 891 patients with documented PAD and 777 control subjects. FGG genotypes were determined by exonuclease (TaqMan) assays. FGG genotype frequencies were not significantly different between PAD patients (CC: 57.3%, CT: 36.7%, TT: 5.8%) and control subjects (CC: 60.9%, CT: 33.5%, TT 5.6%; p = 0.35). In a multivariate logistic regression analysis including age, sex, smoking, diabetes, arterial hypertension and hypercholesterolemia, the FGG 10034 T variant was not significantly associated with the presence of PAD (Odds ratio 1.07, 95% confidence interval 0.84 - 1.37; p = 0.60). The FGG 10034C > T polymorphism was furthermore not associated with age at onset of PAD. We conclude that the thrombophilic FGG 10034 T gene variant does not contribute to the genetic susceptibility to PAD.

Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin

AbstractActivated platelets secrete a negatively charged polymer, polyphosphate (polyP). Here, we explore the interactions of polyP with fibrin(ogen) and its effect on fibrin structure and fibrinolysis. Electrophoretic mobility and binding assays indicate that polyP interacts with fibrinogen and soluble fibrin. Clots formed in the presence of polyP exhibited reduced turbidity and permeability indicative of a tighter fibrin network, but these changes were not related to cross-linking or fibrinopeptide release. Microscopy showed a change in fibrin distribution in clots formed with polyP; with formation of tight aggregates of fibrin fibers interspaced with large pores in contrast to homogenous fiber distribution in control clots. Lysis by tissue plasminogen activator (tPA) and plasminogen or plasmin was delayed in clots formed with polyP and depended on both the activator and polyP concentration. Adding polyP to the clot after fibrin formation or to repolymerizing soluble fibrin did not affect lysis, indicating changes induced by polyP occur at the level of conversion of fibrinogen to fibrin. Surface plasmon resonance showed that the presence of polyP reduced the binding of both plasminogen and tPA to partially lysed fibrin surfaces. These data show that polyP directly influences fibrin architecture and attenuates fibrinolysis through reduced binding of fibrinolytic proteins.

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion.

Metabolic signaling in aging and disease

The rate of biosynthesis and turnover of plasma protein fibrinogen is a marker of metabolic signaling in aging and disease. The rate in the young normal human subject of 0.260 mg/ml/24 hours increases to 0.378 in older normal subjects and to 0.466 in age matched coronary thrombosis patients measured by endogenous labeling of fibrinogen with L‐glutamic acid‐C14. This increased rate of fibrinogen turnover was traced to generation of occlusive fibrin polymers labeled by glycine‐C14 ethyl esters in the presence of active fibrin stabilizing factor. Circulating fibrin increased 520% (P<0.001) above normal in ischemic thrombotic cerebrovascular disease. Long chain saturated free fatty acids (FFA) exert not only primary control over incorporation of C14 amino acids into the fibrinogen structure but also activate the cascade sequence of reactions which convert fibrinogen into occlusive fibrin. FFA are normally transported by albumin to sites of energy metabolism. Decline in albumin synthesis in aging causes an increase in unbound FFA which induce not only increased fibrinogen synthesis but also activation of Hageman Factor generation of thrombin conversion of fibrinogen into occlusive fibrin. It has now been discovered that induction of a normal FFA/albumin ratio in plasma of coronary thrombosis and thrombotic stroke patients regenerates a normal level of fibrinogen synthesis by human hepatocytes.

Anticoagulant Therapy for Percutaneous Coronary Intervention

Received June 2, 2009; accepted November 16, 2009. Percutaneous coronary intervention (PCI) is the most commonly performed invasive therapeutic cardiac procedure and plays an important role in the treatment of ischemic heart disease. Since the first description of coronary angioplasty in a human by Gruntzig,1 the technique, equipment, and associated pharmacotherapy have undergone substantial evolution, leading to significant improvements in periprocedural complications.2 In particular, procedural anticoagulant therapy has been the focus of numerous clinical trials, and several options are now available and supported by practice guidelines; each agent has both advantages and disadvantages, and procedural pharmacotherapy continues to be a focus of drug development. The purpose of this review is to summarize the goals of anticoagulant therapy during PCI, the pharmacokinetics and pharmacodynamics of available agents, and the clinical data surrounding each agent and to identify new agents in development. The goals of pharmacotherapy during PCI are 2-fold: (1) to mitigate the sequelae of iatrogenic plaque rupture from balloon angioplasty or stenting and (2) to reduce the risk of thrombus formation on intravascular PCI equipment. Central to these thrombotic events is thrombin (factor IIa). Iatrogenic damage to the endothelium during PCI leads to increased expression of tissue factor, activation of the coagulation cascade, and formation of activated factor Xa. This ultimately leads to the generation of thrombin, conversion of fibrinogen to fibrin, and thrombus formation.3 In addition to its effects on fibrin, thrombin also directly activates platelets, enhances platelet aggregation, and is proinflammatory.4 Because of its multiple actions in promoting thrombosis, the focus of most anticoagulant agents is thrombin inhibition. Available agents for use include unfractionated heparin (UFH), low-molecular-weight heparins (LMWH, of which enoxaparin has the largest body of clinical data), the synthetic pentasaccharides (of which fondaparinux has the largest body of clinical data), and the …

Fibrinogen Regulates the Cytotoxicity of Mycobacterial Trehalose Dimycolate but Is Not Required for Cell Recruitment, Cytokine Response, or Control of Mycobacterial Infection

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6'-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was adsorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in an in vivo model of mycobacterial granuloma formation and in infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production in response to trehalose dimycolate, nor was there a difference in lung histopathology or overall bacterial burden in mice infected with Mycobacterium tuberculosis. In this model, fibrin(ogen) was not required for cell recruitment, cytokine response, or response to infection, but it promoted granulation tissue formation and suppressed leukocyte necrosis.

Structure‐Based Design of Residue 1 Analogs of the Direct Thrombin Inhibitor Pentapeptide FM 19

Myocardial ischemia and other acute coronary syndromes are leading causes of death worldwide, and often result from a thrombus that blocks an atherosclerotic coronary artery. A key enzyme in thrombus formation is the serine protease thrombin, which is responsible for both the conversion of soluble fibrinogen into insoluble fibrin, as well as the activation of the GPCRs, PAR1 and PAR4, which stimulate platelet aggregation. Thus, thrombin is an attractive target for anticoagulant and antithrombotic therapy. Previous studies in our laboratory led to the development of lead compound FM 19 (D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH2), which shows modest potency as a thrombin inhibitor. The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed potential sites for modification to improve potency. This study reports replacements to the first residue (D-Arg1) of FM 19, which seek to improve potency by removing the N-terminal amine to eliminate an adverse electrostatic interaction, and alterations to the length of the side chain to eliminate an unfavorable eclipsed conformation observed in the X-ray structure. This study produced two compounds, 1 and 9, with improved alpha-thrombin inhibition (IC50 values of 0.66 +/- 0.20 microM and 0.57 +/- 0.12 microM, respectively).

Engineering fibrin matrices: The engagement of polymerization pockets through fibrin knob technology for the delivery and retention of therapeutic proteins

Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob–protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants ( K D) obtained using surface plasmon resonance indicate that these fusions have μ m binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob:pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.

Study on the debridement efficacy of formulated enzymatic wound debriding agents by in vitro assessment using artificial wound eschar and by an in vivo pig model

An in vitro efficacy study using newly developed artificial wound eschar (AWE) substrate was conducted for assessing enzyme dose response. The AWE substrate is prepared by the enzymatic conversion of fibrinogen to fibrin in the presence of collagen, fibrin, and elastin to form an insoluble planar matrix. AWE substrate was placed on Franz Diffusion Cells for continuously monitoring the debridement progress. A parallel in vivo study was performed using pig thermal-burn wounds. Papain at concentrations of 200, 400, 800, and 1,600 U/mg was used as the model debriding enzyme for both studies. The data from the first 5 hours of the in vitro testing showed that debriding activity increased as the enzyme concentration increased. The histological results of the in vivo biopsy samples showed that enzyme doses above 800 and 1,600 U/mg successfully achieved debridement on day 8, while lower treatment groups still contained eschar tissue. Using the histological measurement results (wound depth score) a dose response that correlated to the in vitro assessment was found. Granulation tissue maturity and reepithelialization displayed correlation with the enzyme dose. Results indicate that AWE substrate can be used to predict debridement efficacy in vitro when correlation to the in vivo assessment is achieved.

Activation of NFkB and coagulation in lung injury by hyperoxia and excessive mechanical ventilation: one more reason “low and slow” is the way to go?

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Peak thrombin generation and subsequent venous thromboembolism: the Longitudinal Investigation of Thromboembolism Etiology (LITE) study.

Thrombin is an enzyme that is essential for the acceleration of the coagulation cascade and the conversion of fibrinogen to clottable fibrin. We evaluated the relationship of basal peak thrombin generation with the risk of future venous thromboembolism (VTE), and determined whether associations were independent of other coagulation markers. The Longitudinal Investigation of Thromboembolism Etiology (LITE) study investigated VTE in two prospective population-based cohorts: the Atherosclerosis Risk in Communities (ARIC) study and the Cardiovascular Health Study (CHS). Peak thrombin generation was measured on stored plasma in a nested case-control sample (434 cases and 1004 controls). Logistic regression was used to estimate the relationship of peak thrombin generation with VTE, adjusted for age, sex, race, center, and body mass index. Mediation was evaluated by additionally adjusting for factor VIII and D-dimer. Relative to the first quartile of peak thrombin generation, the odds ratio (OR) of VTE for those above the median was 1.74 [95% confidence interval (CI) 1.28-2.37]. The association was modestly attenuated by adjustment for FVIII and D-dimer (OR 1.47, 95% CI 1.05-2.05). Associations appeared to be stronger for idiopathic than for secondary VTE. Elevated peak thrombin generation more than added to the VTE risk associated with FV Leiden or low activated partial thromboplastin time. In this prospective study of two independent cohorts, elevated basal peak thrombin generation was associated with subsequent risk of VTE, independently of established VTE risk factors.