Fibrinogen Catabolism Study of Three Patients with Bacterial Endocarditis and Renal Disease

Three patients with bacterial endocarditis who developed renal failure have been studied by means of a radiofibrinogen catabolism study. Two were studied at the time of bacteraemia and one during the subsequent “immunological phase” of circulating immune complexes. Accelerated fibrinogen catabolism was found in all cases. Intravascular coagulation may be an important factor in the pathogenesis of proliferative glomerulonephritis in such cases.     

Liver Transplantation in Man—IV,Haemorrhage and Thrombosis

Blood coagulation and fibrinolytic factors have been measured in 13 patients treated by liver transplantation. During operation intravascular coagulation and fibrinolysis were increased, but seldom to a degree which would cause abnormal bleeding. Measurement of the catabolism of radioactive fibrinogen showed that increased intravascular coagulation continued for long periods after the operation. Despite secondarily increased fibrinolysis, there was a high incidence of thrombosis. Treatment with anticoagulants or with fibrinolysis inhibitors may be valuable in these patients.     

Addition of Arsenic Trioxide into Induction Regimens Could Not Accelerate Recovery of Abnormality of Coagulation and Fibrinolysis in Patients with Acute Promyelocytic Leukemia

Aim

All-trans retinoic acid combined to anthracycline-based chemotherapy is the standard regimen of acute promyelocytic leukemia. The advent of arsenic trioxide has contributed to improve the anti-leukemic efficacy in acute promyelocytic leukemia. The objectives of the current study were to evaluate if dual induction by all-trans retinoic acid and arsenic trioxide could accelerate the recovery of abnormality of coagulation and fibrinolysis in patients with acute promyelocytic leukemia.

Methods

Retrospective analysis was performed in 103 newly-diagnosed patients with acute promyelocytic leukemia. Hemostatic variables and the consumption of component blood were comparably analyzed among patients treated by different induction regimen with or without arsenic trioxide.

Results

Compared to patients with other subtypes of de novo acute myeloid leukemia, patients with acute promyelocytic leukemia had lower platelet counts and fibrinogen levels, significantly prolonged prothrombin time and elevated D-dimers (P<0.001). Acute promyelocytic leukemia patients with high or intermediate risk prognostic stratification presented lower initial fibrinogen level than that of low-risk group (P<0.05). After induction treatment, abnormal coagulation and fibrinolysis of patients with acute promyelocytic leukemia was significantly improved before day 10. The recovery of abnormal hemostatic variables (platelet, prothrombin time, fibrinogen and D-dimer) was not significantly accelerated after adding arsenic trioxide in induction regimens; and the consumption of transfused component blood (platelet and plasma) did not dramatically change either. Acute promyelocytic leukemia patients with high or intermediate risk prognostic stratification had higher platelet transfusion demands than that of low-risk group (P<0.05).

Conclusions

Unexpectedly, adding arsenic trioxide could not accelerate the recovery of abnormality of coagulation and fibrinolysis in acute promyelocytic leukemia patients who received all-trans retinoic acid combining chemotherapy.     

Glycolaldehyde induces fibrinogen post-translational modification, delay in clotting and resistance to enzymatic digestion

Glycolaldehyde (GA) is a highly reactive aldehyde that can be generated during inflammation and hyperglycemia. It can react with arginine and lysine residues impairing protein function. As inflammation and diabetes present haemostatic dysfunction, we hypothesized that GA could participate in this process. The aim of this study was to investigate if plasma incubated in the presence of GA presents alteration in the coagulation process. We also aimed to evaluate the role of fibrinogen in GA-induced haemostatic dysfunction. For this purpose, plasma and fibrinogen were each incubated separately, either in the presence or absence of 1 mM GA for 8 and 4 h, respectively. After that, plasma coagulation and fibrin polymerization kinetics were recorded, as well as the kinetic of plasma clot digestion and fibrinolysis protein carbonylation was quantified. An SDS-PAGE was run to check the presence of cross-linking between fibrinogen chains. GA induced a delay in plasma coagulation and in fibrin polymerization. Maximum absorbance decreased after GA treatment, indicating the generation of thinner fibers. Fibrin generated after complete coagulation showed resistance to enzymatic digestion, which could be related to the generation of thinner fibers. Protein carbonylation also increased after GA treatment. All parameters could be reversed with AMG (a carbonyl trap) co-treatment. The data presented herein indicate that GA causes post-translational modification of lysine and arginine residues, which are central to many events involving fibrinogen to fibrin conversion, as well as to fibrinolysis. These modifications lead to the generation of persistent clots and may contribute to mortality seen in pathologies such diabetes and sepsis.     

Modern Aspects of the Treatment of Hemophilia

The prognosis of hemophilic hemorrhages is greatly dependent on the therapy which is administered during the first three to five days. To prevent overloading of the circulation, highly active preparations of the antihemophilic factor should be available.A survey of the recently developed concentrated products of this factor and of their suitability for clinical use is presented. The authors describe their own experiences in the treatment of a number of patients with hemophilia A by administration of so-called “two-donor” fibrinogen.The effect of this “two-donor” fibrinogen was not inferior to results obtained else-where with the so-called I-O product, prepared from a much greater quantity of blood plasma.     

Discrepant Fibrinolytic Response in Plasma and Whole Blood during Experimental Endotoxemia in Healthy Volunteers

Background

Sepsis induces early activation of coagulation and fibrinolysis followed by late fibrinolytic shutdown and progressive endothelial damage. The aim of the present study was to investigate and compare the functional hemostatic response in whole blood and plasma during experimental human endotoxemia by the platelet function analyzer, Multiplate and by standard and modified thrombelastography (TEG).

Methods

Prospective physiologic study of nine healthy male volunteers undergoing endotoxemia by means of a 4-hour infusion of E. coli lipopolysaccharide (LPS, 0.5 ng/kg/hour), with blood sampled at baseline and at 4 h and 6 h. Physiological and standard biochemical data and coagulation tests, TEG (whole blood: TEG, heparinase-TEG, Functional Fibrinogen; plasma: TEG±tissue-type plasminogen activator (tPA)) and Multiplate (TRAPtest, ADPtest, ASPItest, COLtest) were recorded. Mixed models with Tukey post hoc tests and correlations were applied.

Results

Endotoxemia induced acute SIRS with increased HR, temperature, WBC, CRP and procalcitonin and decreased blood pressure. It also induced a hemostatic response with platelet consumption and reduced APTT while INR increased (all p<0.05). Platelet aggregation decreased (all tests, p<0.05), whereas TEG whole blood clot firmness increased (G, p = 0.05). Furthermore, during endotoxemia (4 h), whole blood fibrinolysis increased (clot lysis time (CLT), p<0.001) and Functional Fibrinogen clot strength decreased (p = 0.049). After endotoxemia (6 h), whole blood fibrinolysis was reduced (CLT, p<0.05). In contrast to findings in whole blood, the plasma fibrin clot became progressively more resistant towards tPA-induced fibrinolysis at both 4 h and 6 h (p<0.001).

Conclusions

Endotoxemia induced a hemostatic response with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by downregulation of fibrinolysis, with a discrepant fibrinolytic response in plasma and whole blood. The finding that blood cells are critically involved in the vasculo-fibrinolytic response to acute inflammation is important given that disturbances in the vascular system contribute significantly to morbidity and mortality in critically ill patients.     

Obesity-related derangements of coagulation and fibrinolysis: a study of obesity-discordant monozygotic twin pairs

Coagulation and fibrinolytic activities are under strong genetic control. We studied the effects of acquired obesity, independent of genetic factors on coagulation and fibrinolysis activities in obesity-discordant healthy monozygotic (MZ) twin pairs. Fourteen obesity-discordant (BMI within-pair difference >3 kg/m(2)) and 10 concordant (BMI difference <2 kg/m(2)) MZ twin pairs were identified from the nationwide FinnTwin16 study. Body composition (dual-energy x-ray absorptiometry), abdominal fat distribution (magnetic resonance imaging), liver fat (magnetic resonance spectroscopy), high sensitivity C-reactive protein, insulin sensitivity (euglycemic hyperinsulinemic clamp), and a panel of different markers of blood coagulation and fibrinolysis in the fasting state were measured. Strong resemblance was observed in most coagulation factors within all twin pairs, with the intraclass correlations ranging from 0.73 to 0.97, P < 0.03. However, the activities of fibrinogen and FIX, FXI, and FXII, and plasminogen activator inhibitor-1 (PAI-1) activities were increased in the obese co-twins (P < 0.05) and strongly correlated with the measures of adiposity, inflammation, and insulin resistance (r = 0.32-0.73, P < 0.05) among the twin individuals. Intrapair differences in fibrinogen and PAI-1 correlated with those in BMI, adiposity, and fasting insulin levels (r = 0.40-0.58, P < 0.05) indicating the independent effect of obesity. Derangements of blood coagulation and fibrinolysis are present already in early adulthood in obese subjects. Acquired obesity, independent of genetic factors, increases the activities of fibrinogen and activities of FIX, FXI, FXII, and PAI-1. This study confirms the mechanisms of simultaneous activities of intrinsic coagulation factors and impaired fibrinolysis predisposing obese subjects to thrombosis.     

Citrullinated fibrinogen inhibits thrombin-catalysed fibrin polymerization

Citrullination is the post-translational modification of arginine residues by peptidylarginine deiminases (PADIs). Fibrinogen is one substrate of PADIs under physiological conditions. Fibrinogen is an important factor for blood coagulation and inducing inflammation. The citrullinated form of fibrinogen appears in rheumatoid arthritis synovial tissue together with the production of autoantibodies that target self-peptides containing citrulline. However, whether the function of fibrinogen changes after citrullination remains unclear. We found that citrullinated fibrinogen markedly impairs the function of thrombin-catalysed fibrin polymerization and also inhibits fibrin formation. Increased citrullinated fibrinogen might thus affect the balance between coagulation and fibrinolysis and alter antigenicity under physiological conditions. These data suggest that citrullination of proteins could physiologically change functions and subsequently generate pro-inflammatory conditions and autoimmune reactions.     

Modern Aspects of the Treatment of Hemophilia. II

Ways of obtaining “two-donor” fibrinogen, as described in an earlier communication,¹ with an optimal and more constant amount of Factor VIII activity were investigated. Four modifications of the method as originally described by Nitschmann et al. are described. This freshly prepared modified “two-donor” fibrinogen was used in the treatment of severe or moderately severe hemorrhage in 20 hemophiliacs. Comparison of the results with those obtained by intermittent transfusions of fresh plasma or fresh blood indicated that the modified “two-donor” fibrinogen was definitely the preferable preparation.     

Coagulation and fibrinolysis in intact hydatidiform molar pregnancy.

Tests of coagulation, fibrinolysis, and platelet function were performed in 17 patients with intact molar pregnancies. Women with intact molar pregnancies had higher fibrinogen factor VIII, and fibrinogen degradation products, concentrations and lower prothrombin, factor X, plasminogen, and plasminogen activator concentrations than controls with normal pregnancies. They also had reduced platelet counts and thromboelastographic values, which indicated hypocoagulability. These results suggest that intravascular coagulation occurs in intact hydatidiform molar pregnancies.     

Increased Plasma Clot Permeability and Susceptibility to Lysis Are Associated with Heavy Menstrual Bleeding of Unknown Cause: A Case-Control Study

Background

Formation of compact and poorly lysable clots has been reported in thromboembolic disorders. Little is known about clot properties in bleeding disorders.

Objectives

We hypothesized that more permeable and lysis-sensitive fibrin clots can be detected in women with heavy menstrual bleeding (HMB).

Methods

We studied 52 women with HMB of unknown cause and 52 age-matched control women. Plasma clot permeability (K_s), turbidity and efficiency of fibrinolysis, together with coagulation factors, fibrinolysis proteins, and platelet aggregation were measured.

Results

Women with HMB formed looser plasma fibrin clots (+16% [95%CI 7–18%] Ks) that displayed lower maximum absorbancy (-7% [95%CI -9 – -1%] ΔAbs_max), and shorter clot lysis time (-17% [95%CI -23 – -11%] CLT). The HMB patients and controls did not differ with regard to coagulation factors, fibrinogen, von Willebrand antigen, thrombin generation markers and the proportion of subjects with defective platelet aggregation. The patients had lower platelet count (-12% [95%CI -19 – -2%]), tissue plasminogen activator antigen (-39% [95%CI -41 – -29%] tPA:Ag), and plasminogen activator inhibitor-1 antigen (-28% [95%CI -38 – -18%] PAI-1:Ag) compared with the controls. Multiple regression analysis upon adjustment for age, body mass index, glucose, and fibrinogen showed that decreased tPA:Ag and shortened CLT were the independent predictors of HMB.

Conclusions

Increased clot permeability and susceptibility to fibrinolysis are associated with HMB, suggesting that altered plasma fibrin clot properties might contribute to bleeding disorders of unknown origin.     

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.     

Effect of lectin-binding to fibrinogen D and E domains in coagulation and fibrinolysis

The effect of fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: and inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both γ-γ and α-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Near 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Fibrinogen.

Fibrinogen is a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, fibrinogen is cleaved by thrombin to form fibrin which is the most abundant component of blood clots. As well as controlling blood loss at sites of tissue damage, other properties of fibrinogen have recently been discovered. For example, various cleavage products of fibrinogen and fibrin, released during coagulation and fibrinolysis, respectively, regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types including fibroblasts, endothelial and smooth muscle cells. Current research aims to define the bioactive fibrinogen molecule moieties and cellular receptors involved in these processes. Future studies may provide us with new opportunities to develop agents which are useful in promoting tissue repair or conversely in inhibiting fibrosis in inflammatory and fibroproliferative diseases where endothelial cell damage or chronic leakage of blood proteins is a feature.     

New strategies in the determination of fibrin and fibrin(ogen) derivatives by monoclonal antibodies

Until recently only tests with a limited specificity were available for the assessment of the products of activated coagulation and/or fibrinolysis. Those assays were based on polyclonal antibodies, which crossreact with fibrinogen, and as a consequence they were performed on serum samples i.e. after removal of fibrinogen by clotting. Serum preparation, however, is a notorious source of artefactually high or low levels of fibrin(ogen) degradation products, and is not suitable for the determination of coagulation products. Recently, highly specific monoclonal antibodies (MoAb's) have been developed, the majority of which do not crossreact with fibrinogen. This has enabled new strategies to be developed, i.e. assays using these MoAb's on plasma samples. Furthermore, the new assays can discriminate between (individual) fibrin and fibrinogen degradation products, and coagulation products can be assessed in the same plasma samples.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Effect of Mild Hypothermia on the Coagulation-Fibrinolysis System and Physiological Anticoagulants after Cardiopulmonary Resuscitation in a Porcine Model

The aim of this study was to evaluate the effect of mild hypothermia on the coagulation-fibrinolysis system and physiological anticoagulants after cardiopulmonary resuscitation (CPR). A total of 20 male Wuzhishan miniature pigs underwent 8 min of untreated ventricular fibrillation and CPR. Of these, 16 were successfully resuscitated and were randomized into the mild hypothermia group (MH, n = 8) or the control normothermia group (CN, n = 8). Mild hypothermia (33°C) was induced intravascularly, and this temperature was maintained for 12 h before pigs were actively rewarmed. The CN group received normothermic post-cardiac arrest (CA) care for 72 h. Four animals were in the sham operation group (SO). Blood samples were taken at baseline, and 0.5, 6, 12, 24, and 72 h after ROSC. Whole-body mild hypothermia impaired blood coagulation during cooling, but attenuated blood coagulation impairment at 72 h after ROSC. Mild hypothermia also increased serum levels of physiological anticoagulants, such as PRO C and AT-III during cooling and after rewarming, decreased EPCR and TFPI levels during cooling but not after rewarming, and inhibited fibrinolysis and platelet activation during cooling and after rewarming. Finally, mild hypothermia did not affect coagulation-fibrinolysis, physiological anticoagulants, or platelet activation during rewarming. Thus, our findings indicate that mild hypothermia exerted an anticoagulant effect during cooling, which may have inhibitory effects on microthrombus formation. Furthermore, mild hypothermia inhibited fibrinolysis and platelet activation during cooling and attenuated blood coagulation impairment after rewarming. Slow rewarming had no obvious adverse effects on blood coagulation.     

Thrombolytic properties of an inactive proenzyme form of human urokinase secreted from human kidney cells

The relative fibrin-binding, fibrinolytic and fibrinogenolytic properties of single-chain pro-urokinase, an inactive proenzyme form of human urokinase purified from cultured human kidney cells, and urokinase were compared. The affinity of single-chain pro-urokinase for fibrin was much higher than that of urokinase. In Vitro thrombolytic studies showed that single-chain pro-urokinase is approximately three times more potent in fibrinolysis than urokinase and that it does not degrade fibrinogen in the plasma at a concentration, at which complete plasma clot lysis takes place; whereas, urokinase extensively degrades the fibrinogen in the plasma. These specific, potent thrombolytic properties of single-chain pro-urokinase seem to be due to its high affinity for fibrin and to its conversion from the inactive single-chain form to the active two-chain form on the thrombus by the catalytic amount of plasmin generated during coagulation. This single-chain pro-urokinase obtained from human kidney cells by tissue culture should prove advantageous than urokinase in thrombolytic therapy.     

Homology modeling and structural validation of tissue factor pathway inhibitor

Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either “Tissue Dependent Pathway” (also known as extrinsic pathway) or by “contact activation pathway” (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it''s functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism.