Plasma Fibrinolysis Research Articles

Nitrite Regulation Of Platelet Reactivity Under Physiological Conditions

Nitric oxide (NO) is generated by serial reduction pathway of nitrate and nitrite or by activity of endogenous nitric oxide synthase (NOS), and modulates platelet function and hemostasis. We have shown by aggregometry and flow cytometric analysis of P-selectin that nitrite in the presence of erythrocytes inhibits platelet aggregation and activation after its reduction to NO at low oxygen tension [PLoS One. 2012; 7: e30380. 10.1371/journal.pone.0030380]. We then investigated how nitrite may affect overall clotting processes by regulating platelet function using thrombelastography (TEG), a method used to assess platelet function, fibrin clot formation, and fibrinolysis in blood and/or plasma. We measured the effect of NO donors and nitrite on three TEG parameters, reaction time (R, time to initial fibrin formation), α angle (velocity of clot growth) and maximum amplitude (MA, clot strength), in healthy volunteers. The NO donor (DEANONOate) inhibited all three TEG parameters in response to two independent platelet activators (ADP and AA), in platelet rich plasma (PRP) or whole blood diluted with plasma to yield 20% hematocrit (Hct) to model blood in the microcirculation in vivo. At DEANONOate concentrations ranging from 0-1 μM, R values were progressively prolonged from 1.9 to 3.6 (p=0.008), α angle was decreased from 17.8 to 9.5 (p=0.01) and maximum clot amplitude was reduced from 9.0 to 4.4 (p=0.001) in 20% Hct with ADP stimulation. In contrast, nitrite did not affect clotting parameters in PRP, but exhibited moderate inhibitory effects in 20% Hct at concentrations from 0-10 μM; R values were slightly prolonged from 1.6 to 2.7 (p=0.12), α angle was decreased from 21.8 to 12.8 (p=0.07) and maximum clot amplitude was reduced from 11.0 to 5.0 (p=0.02). This inhibitory effect of nitrite on clotting was greatly enhanced under hypoxic conditions (blood pO2 46.5±11.6 mmHg); R values from 1.4 to 4.0 (p=0.003), α angle from 21.7 to 7.8 (p=0.002), and maximum clot amplitude from 10.0 to 3.4 (p=0.0003). These results suggest that the nitrite effect may be greatest in the microcirculation and be important in differences between arterial and venous clotting. In conclusion, our results show TEG parameters indicate NO inhibition of platelet-mediated blood clotting and that the physiological effect of factors which determine NO bioavailability, such as reduction of blood and tissue nitrite, could be used to predict hemostasis. Disclosures: Schechter: National Institutes of Health: Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases., Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. Patents & Royalties.

The Effective Concentration of Tranexamic Acid for Inhibition of Fibrinolysis in Neonatal Plasma In Vitro

Neonates are at high risk for bleeding complications after cardiovascular surgery. Activation of intravascular fibrinolysis is one of the principal effects of cardiopulmonary bypass that causes poor postoperative hemostasis. Antifibrinolytic medications such as tranexamic acid are often used as prophylaxis against fibrinolysis, but concentration/effect data to guide dosing are sparse for adults and have not been published for neonates. Higher concentrations of tranexamic acid than those necessary for inhibition of fibrinolysis may have adverse effects. Therefore, we investigated the concentration of tranexamic acid necessary to inhibit activated fibrinolysis in neonatal plasma. We conducted an in vitro study using neonatal plasma derived from the placenta/cord units from 20 term, elective cesarean deliveries. Graded concentrations of tranexamic acid were added to aliquots of the pooled plasma before maximally activating fibrinolysis with high-dose tissue-type plasminogen activator. Thromboelastography was then performed with the primary outcome variable being lysis at 30 minutes. These procedures were repeated on pooled adult normal plasma and dilutions of neonatal plasma. The minimum concentrations of tranexamic acid to completely prevent fibrinolysis were 6.54 μg/mL (95% confidence interval, 5.19-7.91) for neonatal plasma and 17.5 μg/mL (95% confidence interval, 14.59-20.41) for adult plasma. Neonatal plasma requires a significantly lower concentration than adult plasma (P < 0.0001, 2-sided Wald test). Our data establish the minimal effective concentration of tranexamic acid necessary to completely prevent fibrinolysis in neonatal plasma in vitro. These data may be useful in designing a dosing scheme for tranexamic acid appropriate for neonates.

Stability of Tranexamic Acid after 12-Week Storage at Temperatures from –20°C to 50°C

Background. Tranexamic acid (TXA) is an antifibrinolytic agent that reduces blood loss during surgery, decreases mortality in civilian and military trauma populations, was adopted for prehospital use by the British military, and is now issued to U.S. Special Operations Forces for use on the battlefield. Objective. This study tested whether storage of TXA ampoules at four temperatures (–20°C, 4°C, 22°C, or 50°C) for 1, 2, 4, and 12 weeks would result in chemical degradation and the loss of activity to block streptokinase-induced fibrinolysis in human plasma. Methods. For each temperature and storage duration, normal plasma, plasma plus streptokinase (SK) (50 units/mL), and plasma + SK + TXA (0.2 μg/mL, n = 4) were tested for D-dimer (DD), for fibrin degradation products (FDP), by thromboelastography (to measure the units/mL of SK needed to get 100% fibrinolysis at 60 minutes [LY60]), and by high-performance liquid chromatography (HPLC). The results were similar for all temperatures and storage durations, and were therefore combined. Results. Streptokinase led to a rise in LY60, DD, and FDP that was significantly (p < 0.05) attenuated with TXA. The results in the three test conditions were LY60: 0.00% ± 0.00%, 70.52% ± 4.7%, 0.02% ± 0.01%; DD: 0.23 ± 0.1, 205.05 ± 101.59, 0.31 ± 0.01 mg/L; and FDP: <10, >40, and <10 μg/mL, respectively. The HPLC results showed no chemical breakdown of TXA. All TXA glass ampoules at –20°C were cracked by week 1. Conclusions. Except for the finding that TXA ampoules cracked when frozen, this study indicated that the drug remains effective when stored under conditions likely to be encountered in the prehospital environment and outside the manufacturer's recommended temperature range for at least 12 weeks.

Proteolytic modulation of factor Xa–antithrombin complex enhances fibrinolysis in plasma

Our previous work showed that purified coagulation factor Xa (FXa) acquires fibrinolysis cofactor activity after plasmin-mediated cleavage. The predominant functional species is a non-covalent heterodimer of 33 and 13kDa, termed Xa33/13, which has predicted newly exposed C-terminal lysines that are important for tissue plasminogen activator (tPA)-mediated plasminogen activation to plasmin. To provide evidence that this mechanism occurs in a physiological context, here we demonstrated the appearance of Xa33 in clotting plasma by western blot analysis. Since the normal fate of FXa is stable association with antithrombin (AT), an AT western blot was conducted, which revealed a band of ~13kDa higher apparent molecular weight than AT that appeared concurrent to Xa33. Sequencing of purified proteins confirmed the generation of Xa13 covalently bound to AT and Xa33 (Xa33/13-AT) by cleavages at Lys–Met339 and Lys–Asp389. Ligand blots demonstrated 125I-plasminogen binding to the Xa33 subunit of plasmin-generated Xa33/13-AT. Purified XaAT added to plasma that was induced to clot enhanced the rate of tPA-mediated fibrinolysis by ~16-fold. Similarly, purified plasminogen activation by tPA was enhanced by ~16-fold by XaAT. Plasmin cleaves XaAT and exposes plasminogen binding sites at least 10-fold faster than FXa. Here we demonstrate a novel function for AT, which accelerates the modulation of FXa into the fibrinolytic form, Xa33/13. The consequent exposure of C-terminal lysine binding sites essential for plasminogen activation enhances fibrinolysis. These results are consistent with a model where auxiliary cofactors link coagulation to fibrinolysis by priming the accelerating role of fibrin.

Alternative Mechanism of Aspirin in Anti-Thrombotic Therapy: Inhibition of Thrombin Activatable Fibrinolysis Inhibitor

The use of aspirin is widely recommended for the prevention of heart attacks owing to its ability to inhibit platelet activation by irreversibly blocking cyclooxygenase 1. However, aspirin also affects the fibrinolytic and hemostatic pathways by mechanisms that are not well understood, causing severe hemorrhagic complications. Here, we investigated the ability of aspirin and aspirin metabolites to inhibit thrombin-activatable fibrinolysis inhibitor (TAFI), the major inhibitor of plasma fibrinolysis. TAFI is activated via proteolytic cleavage by the thrombin-thrombomodulin complex to TAFIa, a carboxypeptidase B-like enzyme. TAFIa modulates fibrinolysis by removing the C-terminal arginine and lysine residues from partially degraded fibrin, which in turn inhibits the binding of plasminogen to fibrin clots. Aspirin and its major metabolites, salicylic acid, gentisic acid, and salicyluric acid, inhibit TAFIa carboxypeptidase activity. Salicyluric acid effectively blocks activation of TAFI by thrombin-thrombomodulin; however, salicylates do not inhibit carboxypeptidase N or pancreatic carboxypeptidase B. Aspirin and other salicylates accelerated the dissolution of fibrin clots and reduced thrombus formation in an in vitro model of fibrinolysis. Inhibition of TAFI represents a novel hemostatic mechanism that contributes to aspirin’s therapy-associated antithrombotic activity and hemorrhagic complications.

Effects of Aescusan on Fibrinolytic Plasma Activity in Patients with Hypothyroidism

The administration of Aescusan into the complex treatment program of patients with hypothyroidism was found to increase total plasma fibrinolysis almost to the control level, normalize the intensity of non-enzymatic fibrinoly- sis, considerably elevate the enzymatic lysis of fibrine and more effectively, as compared with standard treatment pro- gram, restore normal structure of plasma fibrinolysis.

Changes in coagulation and fibrinolysis in the patients with coronary heart disease in acute period and effect of drug intervention

To investigate the changes in coagulation and fibrinolysis parameters during acute phase of coronary heart disease, and to explore the effects of drug intervention in same period. A prospective study was conducted. The changes in plasma von Willebrand factor (vWF), platelet activating factor (PAF), tissue type plasminogen activator (t-PA) of 110 patients with coronary heart disease [50 patients with acute myocardial infarction (AMI), 35 patients with unstable angina pectoris (UAP), 25 patients with ischemic cardiomyopathy (ICM)] were determined immediately at admission and 14 days after treatment. The indexes of 19 healthy individuals were collected as control. At the same time, randomized and double-blind observation of the changes in plasma coagulation and fibrinolysis system indexes including vWF, PAF and t-PA was made in AMI and UAP patients who received conventional treatment (aspirin + low molecular weight heparin) or combination of conventional treatment and clopidogrel for 14 days. The vWF and PAF levels (nmol/L) of AMI and UAP patients at admission were significantly higher than those in healthy control group [vWF: (202.31 ± 27.38)%, (188.65 ± 31.08)% vs. (120.37 ± 18.79)%; PAF: 50.64 ± 13.25, 48.87 ± 13.24 vs. 15.43 ± 9.27, all P < 0.05], however, t-PA (μg/L) was remarkably lower than that in healthy control group (3.52 ± 1.57, 4.03 ± 2.04 vs. 9.54 ± 1.32, both P < 0.01). After 14 days of treatment, the levels of vWF, PAF, t-PA, fibrinogen (Fib, g/L), D-dimer (mg/L) in plasma of AMI and UAP patients were close to those of healthy control group [vWF: (116.56 ± 26.10)%, (111.28 ± 22.31)% vs. (120.37 ± 18.79)%; PAF: 17.48 ± 9.16, 16.23 ± 9.17 vs. 15.43 ± 9.27; t-PA: 7.91 ± 2.42, 9.01 ± 2.01 vs. 9.54 ± 1.32; Fib: 3.64 ± 0.53, 2.74 ± 0.72 vs. 2.92 ± 0.91; D-dimer: 0.370 ± 0.150, 0.288 ± 0.169 vs. 0.255 ± 0.109, all P>0.05]. However, there was no statistical difference in vWF, PAF, t-PA, Fib and D-dimer levels before and after treatment in ICM group compared with healthy control group [vWF: (124.14 ± 21.17)%, (119.44 ± 26.28)% vs. (120.37 ± 18.79)%; PAF: 15.69 ± 7.14, 14.84 ± 9.16 vs. 15.43 ± 9.27; t-PA: 8.62 ± 2.24, 8.07 ± 2.51 vs. 9.54 ± 1.32; Fib: 3.24 ± 0.74, 3.04 ± 0.77 vs. 2.92 ± 0.91; D-dimer: 0.257 ± 0.132, 0.268 ± 0.117 vs. 0.255 ± 0.109, all P>0.05]. Multiple linear regression analysis showed that, in patients with coronary heart disease, PAF be positively correlated with vWF (r = 0.42, P < 0.01), but negatively correlated with t-PA (r = -0.31, P < 0.01). In patients with AMI and UAP, using the treatment of clopidogrel for 14 days, the levels of vWF, PAF, t-PA, Fib and D-dimer in plasma showed no significant differences compared with those of conventional treatment group [vWF: (120.16 ± 16.57)% vs. (118.12 ± 14.32)%; PAF: 12.01 ± 3.70 vs. 13.33 ± 1.10; t-PA: 9.75 ± 1.60 vs. 7.59 ± 2.46; Fib: 2.73 ± 0.49 vs. 3.09 ± 0.50; D-dimer: 0.233 ± 0.101 vs. 0.252 ± 0.088, all P>0.05]. There were changes in coagulation and fibrinolysis status in patients with AMI and UAP at the initial stage, the level of t-PA was decreased significantly. For patients with AMI and UAP, there were no obvious differences in coagulation and fibrinolysis parameters between combination therapy of clopidogrel and aspirin and aspirin alone.

Maternal Malaria Induces a Procoagulant and Antifibrinolytic State That Is Embryotoxic but Responsive to Anticoagulant Therapy

Low birth weight and fetal loss are commonly attributed to malaria in endemic areas, but the cellular and molecular mechanisms that underlie these poor birth outcomes are incompletely understood. Increasing evidence suggests that dysregulated hemostasis is important in malaria pathogenesis, but its role in placental malaria (PM), characterized by intervillous sequestration of Plasmodium falciparum, proinflammatory responses, and excessive fibrin deposition is not known. To address this question, markers of coagulation and fibrinolysis were assessed in placentae from malaria-exposed primigravid women. PM was associated with significantly elevated placental monocyte and proinflammatory marker levels, enhanced perivillous fibrin deposition, and increased markers of activated coagulation and suppressed fibrinolysis in placental plasma. Submicroscopic PM was not proinflammatory but tended to be procoagulant and antifibrinolytic. Birth weight trended downward in association with placental parasitemia and high fibrin score. To directly assess the importance of coagulation in malaria-induced compromise of pregnancy, Plasmodium chabaudi AS-infected pregnant C57BL/6 mice were treated with the anticoagulant, low molecular weight heparin. Treatment rescued pregnancy at midgestation, with substantially decreased rates of active abortion and reduced placental and embryonic hemorrhage and necrosis relative to untreated animals. Together, the results suggest that dysregulated hemostasis may represent a novel therapeutic target in malaria-compromised pregnancies.

Evidence that fibrinolytic changes in paediatric obesity translate into a hypofibrinolytic state

Paediatric obesity, like adulthood obesity, is associated with an increase of fibrinolysis inhibitors. No study, however, has evaluated the impact of these changes on plasma fibrinolytic capacity. We investigated plasma fibrinolysis and the role therein of the fibrinolytic changes associated with obesity in 59 obese children (body mass index > 95th percentile) and 40 matched controls. Fibrinolysis was investigated by measuring 1) the plasma levels of relevant fibrinolytic factors; 2) the in vitro fibrinolytic capacity under different conditions, using a microplate plasma clot lysis assay; 3) the circulating levels of markers of clotting and fibrinolysis activation. Plasminogen activator inhibitor 1 (PAI-1), total thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen levels were higher in obese children as compared to controls (p<0.01). Plasma clots from obese children lysed significantly slower than control clots when exposed to exogenous plasminogen activator, indicating a greater resistance to fibrinolysis. By the use of a selective inhibitor of activated TAFI and by regression analyses we found that fibrinolysis resistance in obese samples was attributable to PAI-1 increase and to enhanced TAFI activation. The ratio between the circulating levels of D-dimer and thrombin-antithrombin complex, a marker of in vivo fibrinolysis, was significantly lower in obese children, suggesting a reduced fibrinolytic efficiency. These data indicate that paediatric obesity is associated with a hypofibrinolytic state which might contribute to the increased thrombotic risk associated with this condition.

Carbon Monoxide-Releasing Molecule-2 Enhances Coagulation and Diminishes Fibrinolytic Vulnerability in Diluted Plasma In Vitro

A carbon monoxide-releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) enhances coagulation and attenuates vulnerability to fibrinolysis in normal and hemophiliac human plasma. We tested the hypothesis that plasma diluted with resuscitative fluids would demonstrate improved coagulation and decreased fibrinolytic vulnerability after exposure to CORM-2. Normal, platelet-poor plasma was diluted 0%, 20%, 30%, 40%, or 50% with 0.9% NaCl (NS) or low-molecular-weight hydroxyethyl starch (VOL) and, subsequently, exposed to 0 μmol/L or 100 μmol/L CORM-2 before activation with tissue factor (n = 4 per condition). Additional plasma samples diluted with NS or VOL (0% or 30%) were exposed to 0 μmol/L or 100 μmol/L CORM-2 and 0 U/mL or 100 U/mL tissue-type plasminogen activator to assess fibrinolytic vulnerability (n = 8 per condition). Thrombelastographic data were collected until either clot strength stabilized or clot lysis occurred, as appropriate. CORM-2 exposure maintained normal to supranormal velocity of clot formation and strength in plasma diluted up to 40% with NS. In contrast, although CORM-2 exposure improved coagulation kinetics, dilution with VOL markedly degraded thrombus formation kinetics. Similarly, fibrinolytic vulnerability to tissue-type plasminogen activator was markedly improved by CORM-2 exposure in samples diluted with NS, whereas VOL-diluted thrombi were still abnormally weak and easily lysed compared with undiluted samples despite CORM-2 exposure. CORM-2 exposure attenuated the decrease in coagulation kinetics and enhancement of fibrinolytic vulnerability associated with hemodilution. Extensive preclinical investigation remains to be performed to determine the route of administration, safety, and efficacy of CORM-2 and other CORMs to treat trauma-associated bleeding.

Carbon monoxide releasing molecule-2 attenuates the anticoagulant and amplifies the hypofibrinolytic effects of hypothermia in human plasma in vitro

Hypothermia is known to contribute to coagulopathy in trauma and other major surgical procedures. Although the effects of hypothermia on coagulation have been characterized, the effects on fibrinolysis remained to be elucidated. Thus, our goals were to discern the effects of hypothermia on fibrinolysis in human plasma, and secondarily determine if a new procoagulant/antifibrinolytic molecule, carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) would modify thrombus growth/disintegration under hypothermic conditions. Normal plasma was exposed to 0 or 100 μmol/l CORM-2, with coagulation activated by tissue factor and fibrinolysis initiated with 100 U/ml tissue-type plasminogen activator. Plasma samples were exposed to 37°, 35°, 33°, 31°, 29°, or 27°C (n = 6 per temperature/CORM-2 concentration). Thrombus growth/disintegration kinetics were monitored with thrombelastography until clot lysis time occurred. Hypothermia significantly prolonged the onset and decreased the velocity of clot growth in plasma without decreasing clot strength. Although hypothermia did not affect the time to onset of fibrinolysis, it did significantly decrease the velocity of lysis. The addition of CORM-2 significantly increased the velocity of clot growth and clot strength at all temperatures tested compared with unexposed plasma. Further, CORM-2 addition significantly prolonged the onset of fibrinolysis and diminished the velocity of lysis. Hypothermia resulted in slower growing, slower lysing thrombi in normal plasma. CORM-2 enhanced coagulation and markedly attenuated fibrinolysis at all temperatures tested. Further investigation is warranted to determine if CORM-2 administration can improve hemostasis in preclinical models of hypothermia and trauma.

Treatment of episodes of hereditary angioedema with C1 inhibitor: serial assessment of observed abnormalities of the plasma bradykinin-forming pathway and fibrinolysis

Treatment of episodes of hereditary angioedema with C1 inhibitor: serial assessment of observed abnormalities of the plasma bradykinin-forming pathway and fibrinolysis

Carbon Monoxide Releasing Molecule-2 Enhances Coagulation and Diminishes Fibrinolytic Vulnerability in Plasma Exposed to Heparin or Argatroban

It has been recently demonstrated that a carbon monoxide releasing molecule (tricarbonyldichlororuthenium [II] dimer; CORM-2) enhances coagulation and attenuates vulnerability to fibrinolysis in normal and hemophiliac human plasma. We tested the hypothesis that plasma anticoagulated with heparin or argatroban would demonstrate improved coagulation and decreased fibrinolytic vulnerability after exposure to CORM-2. Normal plasma was anticoagulated with 0 to 0.1 U/mL unfractionated heparin or 0 to 1 μg/mL argatroban. Samples were subsequently exposed to 0 or 100 μM CORM-2 and activated with tissue factor. Additional samples with the same anticoagulant and CORM-2 exposure schema were incubated with 100 U/mL tissue-type plasminogen activator (tPA) to assess fibrinolytic vulnerability. Thrombelastographic data were collected until either clot strength stabilized or clot lysis occurred as appropriate. In the absence of tPA, CORM-2 significantly increased the velocity of clot growth in heparin (75%) and argatroban-exposed (40%) samples. Clot strength was also significantly increased in heparin (69%) and argatroban-exposed (72%) samples. In the presence of tPA, CORM-2-treated samples had even greater (94%-731%) increases in velocity of growth and strength after exposure to either anticoagulant and significantly increased clot lysis time (103%-200%). CORM-2 exposure resulted in faster-growing, stronger, longer-lived thrombi after anticoagulation with heparin or argatroban. Additional preclinical investigation is warranted to determine whether CORM-2 administration will be useful in attenuating bleeding complications associated with thromboprophylaxis.

Successful C1 inhibitor short-term prophylaxis during redo mitral valve replacement in a patient with hereditary angioedema

Hereditary angioedema is characterized by sudden episodes of nonpitting edema that cause discomfort and pain. Typically the extremities, genitalia, trunk, gastrointestinal tract, face, and larynx are affected by attacks of swelling. Laryngeal swelling carries significant risk for asphyxiation. The disease results from mutations in the C1 esterase inhibitor gene that cause C1 esterase inhibitor deficiency. Attacks of hereditary angioedema result from contact, complement, and fibrinolytic plasma cascade activation, where C1 esterase inhibitor irreversibly binds substrates. Patients with hereditary angioedema cannot replenish C1 esterase inhibitor levels on pace with its binding. When C1 esterase inhibitor is depleted in these patients, vasoactive plasma cascade products cause swelling attacks. Trauma is a known trigger for hereditary angioedema attacks, and patients have been denied surgical procedures because of this risk. However, uncomplicated surgeries have been reported. Appropriate prophylaxis can reduce peri-operative morbidity in these patients, despite proteolytic cascade and complement activation during surgical trauma. We report a case of successful short-term prophylaxis with C1 esterase inhibitor in a 51-year-old man with hereditary angioedema who underwent redo mitral valve reconstructive surgery.

The Effective Concentration of Epsilon-Aminocaproic Acid for Inhibition of Fibrinolysis in Neonatal Plasma in Vitro

Pediatric patients, particularly neonates, are at high risk for bleeding complications after cardiovascular surgery because of their immature hemostatic system, small size, and the complex operations they require. Activation of intravascular fibrinolysis is one of the principle effects of cardiopulmonary bypass that causes poor postoperative hemostasis. This complication has long been recognized and treated with antifibrinolytic medications, including the lysine analog epsilon aminocaproic acid (EACA). The therapeutic plasma concentration of EACA has been scientifically determined for the adult population, but the current recommended dosage for neonates has been empirically derived from adult studies. Therefore, we investigated the appropriate concentration of EACA for neonates undergoing bypass. We conducted an in vitro study using neonatal plasma derived from the placenta/cord units from 20 term, elective cesarean deliveries. Graded concentrations of EACA were added to aliquots of the plasma pool before activating fibrinolysis with tissue-type plasminogen activator. Standard kaolin-activated thromboelastograms were then run with the primary outcome variable being estimated percent lysis. These procedures were repeated on samples of commercially available pooled adult normal plasma for comparison. We found that neonatal plasma required significantly lower concentrations of EACA to completely prevent fibrinolysis than did adult plasma (44.2 microg/mL and 47.8 microg/mL for neonatal plasma and 94.4 and 131.4 microg/mL in adult plasma for 400 and 1000 U/mL of plasminogen activator, respectively, P < 0.001). Our data establish the minimal effective concentration of EACA necessary to completely prevent fibrinolysis in neonatal blood in vitro. This concentration is significantly less than that targeted by current dosing schemes, indicating that neonates are possibly being exposed to greater levels of EACA than is clinically necessary.

Carbon Monoxide Releasing Molecule-2 Enhances Coagulation and Diminishes Fibrinolytic Vulnerability in Subjects Exposed to Warfarin

Introduction It has been recently demonstrated that a carbon monoxide releasing molecule (tricarbonyldichlororuthenium (II) dimer; CORM-2) enhances coagulation and attenuates vulnerability to fibrinolysis in normal and hemophiliac human plasma. We tested the hypothesis that plasma obtained from warfarin-treated subjects would demonstrate improved coagulation and decreased fibrinolytic vulnerability following exposure to CORM-2. Materials and Methods Anonymous donor plasma samples with international normalized ratios (INR) values ranging from 1.5-5.4 were exposed to 0 or 100 µM CORM-2 and activated with tissue factor (12 samples). Additional samples within the same INR range were exposed to 0 or 100 µM CORM-2 and 0 or 100 U/ml tissue-type plasminogen activator (tPA) to assess fibrinolytic vulnerability (8 samples). Thrombelastographic data were collected until either clot strength stabilized or clot lysis occurred as appropriate. Results and Conclusions In the absence of tPA, all but one sample (INR=1.5) demonstrated a marked increase in the velocity of clot formation (40-577%) and strength (42-180%) following CORM-2 exposure. Of interest, in the presence of tPA, all samples (including the previously unresponsive sample) were noted to have an increase in the velocity of clot formation and strength, coupled with a prolonged onset to maximal rate of clot lysis (60-242%) and increased clot lysis time (74-149%). As with normal and hemophilic plasma, both enhancement of coagulation and attenuation of fibrinolysis occur following CORM-2 exposure in plasma from warfarin-treated subjects. Future investigation must determine if carbon monoxide releasing molecules could be used therapeutically to control bleeding in warfarin-treated patients.

A combined immunostimulatory and immunoinhibitory short interference RNA reduces hypercoagulability in a rat model of acute promyelocytic leukaemia

Acute promyelocytic leukaemia (APL) confers an increased risk of thrombosis and bleeding. Current treatments are insufficient to inhibit these complications. We recently showed that a combined immunoinhibitory and immunostimulatory short interference (si) RNA effectively inhibited leukaemic growth and metastasis in rats with APL. We now asked if the reported anti-leukaemic effects of siRNA treatment could be explained by inhibition of hypercoagulability. We measured markers of coagulation and fibrinolysis in plasma collected from APL rats with overt leukaemia using conventional assays. Coagulopathy developed in untreated leukaemic rats evidenced by increase in several haemostatic markers. Treatment of leukaemic rats with the siRNA reduced (p < 0.05) the concentration of thrombin-anti-thrombin complex (a marker of coagulation) by 40% compared with rats treated with an inactive, control siRNA. Substantial reductions (p < 0.05) were also obtained for two markers of fibrinolysis: D-dimer (72%) and plasminogen activator inhibitor type 1 (51%). The activity of tissue factor, the main initiator of coagulation, was not increased (p > 0.05) in untreated leukaemic rats compared with healthy rats, and did not change (p > 0.05) upon treatment with the siRNA. The bifunctional siRNA reduces the hypercoagulable state in APL in addition to its direct anti-leukaemic properties, supporting testing of this small molecule in human APL.

Carbon monoxide-releasing molecule-2 decreases fibrinolysis in human plasma

Carbon monoxide, derived from carbon monoxide-releasing molecules, has been recently demonstrated to enhance the velocity of formation and strength of plasma thrombi. We tested the hypothesis that carbon monoxide-releasing molecule-2 would modulate fibrinolysis of plasma thrombi. Normal plasma was exposed to 0, 25, 50, 100 or 200 micromol/l carbon monoxide-releasing molecule-2, with coagulation activated with tissue factor and fibrinolysis initiated with tissue-type plasminogen activator. Additional experiments utilized factor XIII, plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor or alpha2-antiplasmin-deficient plasmas. Thrombus growth/disintegration kinetics was monitored with thrombelastography. Carbon monoxide-releasing molecule-2, in a concentration-dependent fashion, increased the velocity of thrombus formation and strength, and markedly attenuated fibrinolysis in normal plasma. In factor XIII-deficient plasma, carbon monoxide-releasing molecule-2 mediated effects on thrombus growth/disintegration kinetics were similar to that seen with normal plasma; however, carbon monoxide-releasing molecule-2 had a less marked effect on thrombus growth/disintegration in both plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor-deficient plasma, with even less carbon monoxide-releasing molecule-2-mediated effects noted in alpha2-antiplasmin-deficient plasma. Carbon monoxide-releasing molecule-2 attenuated fibrinolysis by enhancing the velocity of clot growth and strength while augmenting the effects of plasminogen activator inhibitor-1, thrombin activatable fibrinolysis inhibitor and alpha2-antiplasmin. These findings serve as the rationale for further investigations to determine if carbon monoxide-releasing molecules could be utilized as hemostatic agents.

Tissue factor-expressing monocytes inhibit fibrinolysis through a TAFI-mediated mechanism, and make clots resistant to heparins

Thrombin is the main activator of the fibrinolysis inhibitor TAFI (thrombin activatable fibrinolysis inhibitor) and heightened clotting activation is believed to impair fibrinolysis through the increase of thrombin activatable fibrinolysis inhibitor activation. However, the enhancement of thrombin generation by soluble tissue factor was reported to have no effect on plasma fibrinolysis and it is not known whether the same is true for cell-associated tissue factor. The aim of this study was to evaluate the effect of tissue factor-expressing monocytes on plasma fibrinolysis in vitro. Tissue factor expression by human blood mononuclear cells (MNC) and monocytes was induced by LPS stimulation. Fibrinolysis was spectrophotometrically evaluated by measuring the lysis time of plasma clots containing LPS-stimulated or control cells and a low concentration of exogenous tissue plasminogen activator. LPS-stimulated MNC (LPS-MNC) prolonged fibrinolysis time as compared to unstimulated MNC (C-MNC) in contact-inhibited but not in normal citrated plasma. A significantly prolonged lysis time was observed using as few as 30 activated cells/microL. Fibrinolysis was also impaired when clots were generated on adherent LPS-stimulated monocytes. The antifibrinolytic effect of LPS-MNC or LPS-monocytes was abolished by an anti-tissue factor antibody, by an antibody preventing thrombin-mediated thrombin activatable fibrinolysis inhibitor activation, and by a TAFIa inhibitor (PTCI). Assays of thrombin and TAFIa in contact-inhibited plasma confirmed the greater generation of these enzymes in the presence of LPS-MNC. Finally, the profibrinolytic effect of unfractionated heparin and enoxaparin was markedly lower (approximately 50%) in the presence of LPS-MNC than in the presence of a thromboplastin preparation displaying an identical tissue factor activity. Our data indicate that LPS-stimulated monocytes inhibit fibrinolysis through a tissue factor-mediated enhancement of thrombin activatable fibrinolysis inhibitor activation and make clots resistant to the profibrinolytic activity of heparins, thus providing an additional mechanism whereby tissue factor-expressing monocytes/macrophages may favor fibrin accumulation and diminish the antithrombotic efficacy of heparins.

Infusion of C1 Inhibitor as Therapy for Swelling in Hereditary Angioedema Patients Reverses Abnormalities of the Plasma Bradykinin-forming Pathway and Fibrinolysis

Infusion of C1 Inhibitor as Therapy for Swelling in Hereditary Angioedema Patients Reverses Abnormalities of the Plasma Bradykinin-forming Pathway and Fibrinolysis