Fibrinogen depletion after plasma-dilution: impairment of proteolytic resistance and reversal via clotting factor concentrates

SummaryIn trauma patients, resuscitation treatment of intravascular volume may cause haemodilution including blood cell- and plasma-dilution. After plasma-dilution, fibrinogen is the first factor that decreases to critically low concentrations. Fibrin formed in lowered levels is susceptible to fibrinolysis, a natural forerunner for bleeding. To assess whether a fibrinogen concentrate or a factor XIII (FXIII) concentrate can reverse the impairment of fibrin properties after plasma dilution, different laboratory methods were used to determine thrombin generation and fibrin quantity/quality in a normal plasma sample diluted in vitro. Coagulation and clot lysis by plasmin were triggered with tissue factor and rt-PA, respectively. We found that while the endogenous thrombin potential (ETP) was unaffected after plasma-dilution due to postponement of thrombin decay, levels of fibrinogen and hence fibrin were decreased in dilution degree-dependency. The imbalance between influence of the dilution on thrombin activity and fibrin formation brought unexpected outcomes of fibrin properties: the formed clots favoured the degradation by plasmin but the fibrin networks remained tighter/less permeable. This proteolytic tendency was partly overturned by the fibrinogen concentrate added (total fibrinogen ≥ 2 g/l), and much more affected if used in combination with tranexamic acid (a fibrinolysis inhibitor) at small doses. No reversal effect resulted from the FXIII concentrate added. We conclude that plasma-dilution did reduce the proteolytic resistance of formed clots. The fibrinogen concentrate, better together with small doses of tranexamic acid, may reverse the impairment of fibrin property. The FXIII concentrate is not effective in this regard in our in vitro model using platelet-poor plasma.

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Effects of fibrinogen concentrate, factor XIII, and thrombin-activatable fibrinolysis inhibitor on clot firmness and fibrinolytic resistance in the model of hyperfibrinolysis

ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia» ◽

10.25557/igpp.2017.4.8522 ◽

2017 ◽

pp. 44-50

Author(s):

И.А. Будник ◽

О.Л. Морозова ◽

А.А. Цымбал ◽

Б. Шенкман ◽

Ю. Эйнав

Keyword(s):

Factor Xiii ◽

Onset Time ◽

Clot Formation ◽

Clot Lysis ◽

Fibrinogen Concentrate ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Antifibrinolytic Agents ◽

Fibrinolysis Inhibitor ◽

Vitro Model

Цель исследования - изучение возможности коррекции формирования кровяного сгустка и его фибринолитической устойчивости с помощью концентратов фибриногена, фактора XIII и активируемого тромбином ингибитора фибринолиза (TAFI) в модели гиперфибринолиза in vitro . Методика. В образцы цитратной крови, полученной от 24 взрослых здоровых добровольцев, добавляли концентрат фибриногена, фактора XIII и/или TAFI. Фибринолиз индуцировали добавлением тканевого активатора плазминогена. Свертывание крови индуцировали рекальцификацией и добавлением препарата тканевого фактора. Формирование и лизис сгустка изучали методом ротационной тромбоэластометрии. Результаты. Индукция фибринолиза не влияла на время свертывания и скорость формирования сгустка, но значительно уменьшала максимальную плотность сгустка и вызывала его лизис. Концентрат фибриногена замедлял скорость лизиса сгустка; концентрат фактора XIII усиливал механическую прочность сгустка и замедлял скорость его лизиса, не влияя при этом на время начала лизиса; TAFI усиливал механическую прочность и значительно отдалял время начала лизиса, оказывая тем самым наибольший корригирующий эффект. Заключение. Полученные данные демонстрируют потенциальную возможность коррекции гемостатического потенциала крови при гиперфибринолизе с помощью концентратов фибриногена, фактора XIII и TAFI, которые могут стать альтернативой традиционным антифибринолитикам. Aim. To investigate effects of fibrinogen concentrate, factor XIII, and thrombin-activatable fibrinolysis inhibitor (TAFI) on clot formation and fibrinolytic resistance using an in vitro model of hyperfibrinolysis. Methods. Citrated whole blood from 24 adult healthy volunteers was supplemented with fibrinogen concentrate, factor XIII, and/or TAFI. Fibrinolysis was induced by tissue plasminogen activator. Clotting was induced by recalcification and addition of tissue factor and monitored using rotation thromboelastometry. Results. Induction of fibrinolysis did not affect clotting time and the rate of clot formation but significantly reduced the maximum clot firmness and caused lysis of a clot. Addition of fibrinogen concentrate to blood reduced the rate of clot lysis without affecting clot firmness or lysis onset time; addition of factor XIII improved clot firmness and reduced clot lysis rate without affecting lysis onset time; TAFI improved clot firmness and considerably delayed the onset of clot lysis thereby providing the greatest antifibrinolytic effect. Conclusion. Fibrinogen concentrate, factor XIII, and TAFI may potentially serve as an alternative to traditional antifibrinolytic agents and be beneficial for the treatment of patients with hyperfibrinolysis.

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Effect of Heparin on TAFI-Dependent Inhibition of Fibrinolysis: Relative Importance of TAFIa Generated by Clot-Bound and Fluid Phase Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613199 ◽

2002 ◽

Vol 88 (08) ◽

pp. 282-287 ◽

Cited By ~ 14

Author(s):

Anna Pentimone ◽

Bianca Binetti ◽

Marialisa Cramarossa ◽

Donatella Piro ◽

Nicola Semeraro ◽

...

Keyword(s):

Thrombin Generation ◽

Blood Clot ◽

Fluid Phase ◽

Clot Lysis ◽

Relative Importance ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Dependent Inhibition ◽

Fibrinolysis Inhibitor ◽

Dependent Mechanism

SummaryHeparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 µg/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through “localized” TAFIa generation.

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Identification and characterisation of monoclonal antibodies that impair the activation of human thrombin activatable fibrinolysis inhibitor through different mechanisms

Thrombosis and Haemostasis ◽

10.1160/th10-08-0546 ◽

2011 ◽

Vol 106 (07) ◽

pp. 90-101 ◽

Cited By ~ 14

Author(s):

Niraj Mishra ◽

Ellen Vercauteren ◽

Jan Develter ◽

Riet Bammens ◽

Paul J. Declerck ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Clot Lysis ◽

Dependent Manner ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Lysis Time ◽

Human Thrombin ◽

Fibrinolysis Inhibitor ◽

Dose Dependent ◽

Clot Lysis Time

SummaryThrombin activatable fibrinolysis inhibitor (TAFI) forms a molecular link between coagulation and fibrinolysis and is a putative target to develop profibrinolytic drugs. Out of a panel of monoclonal antibodies (MA) raised against TAFI-ACIIYQ, we selected MA-TCK11A9, MA-TCK22G2 and MA-TCK27A4, which revealed high affinity towards human TAFITI- wt. MA-TCK11A9 was able to inhibit mainly plasmin-mediated TAFI activation, MA-TCK22G2 inhibited plasmin- and thrombin-mediated TAFI activation and MA-TCK27A4 inhibited TAFI activation by plasmin, thrombin and thrombin/thrombomodulin (T/TM) in a dose-dependent manner. These MA did not interfere with TAFIa activity. Using an eightfold molar excess of MA over TAFI, all three MA were able to reduce clot lysis time significantly, i.e. in the presence of exogenous TM, MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 47 ± 9.1%, 80 ± 8.6% and 92 ± 14%, respectively, compared to PTCI. This effect was even more pronounced in the absence of TM i.e. MATCK11A9, MA-TCK22G2 and MA-TCK27A4 reduced clot lysis time by 90 ± 14%, 140 ± 12% and 147 ± 29%, respectively, compared to PTCI. Mutagenesis analysis revealed that residues at position 268, 272 and 276 are involved in the binding of MA-TCK11A9, residues 147 and 148 in the binding of MA-TCK22G2 and residue 113 in the binding of MATCK27A4. The present study identified three MA, with distinct epitopes, that impair the activation of human TAFI and demonstrated that MATCK11A9 which mainly impairs plasmin-mediated TAFI activation can also reduce significantly clot lysis time in vitro.

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Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model

Blood ◽

10.1182/blood-2010-08-303677 ◽

2011 ◽

Vol 117 (17) ◽

pp. 4615-4622 ◽

Cited By ~ 28

Author(s):

Ellen Vercauteren ◽

Jan Emmerechts ◽

Miet Peeters ◽

Marc F. Hoylaerts ◽

Paul J. Declerck ◽

...

Keyword(s):

Monoclonal Antibody ◽

Clot Lysis ◽

Fibrin Deposition ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

First Report ◽

Fibrinolysis Inhibitor ◽

Thrombotic Diseases

Abstract The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Activated thrombin activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis and is an attractive target to develop profibrinolytic drugs. TAFI can be activated by thrombin, thrombin/thrombomodulin, or plasmin, but the in vivo physiologic TAFI activator(s) are unknown. Here, we generated and characterized MA-TCK26D6, a monoclonal antibody raised against human TAFI, and examined its profibrinolytic properties in vitro and in vivo. In vitro, MA-TCK26D6 showed a strong profibrinolytic effect caused by inhibition of the plasmin-mediated TAFI activation. In vivo, MA-TCK26D6 significantly decreased fibrin deposition in the lungs of thromboembolism-induced mice. Moreover, in the presence of MA-TCK26D6, plasmin-α2-antiplasmin complexes in plasma of thromboembolism-induced mice were significantly increased compared with a control antibody, indicative of an acceleration of fibrinolysis through MA-TCK26D6. In this study, we show that plasmin is an important TAFI activator that hampers in vitro clot lysis. Furthermore, this is the first report on an anti-TAFI monoclonal antibody that demonstrates a strong profibrinolytic effect in a mouse thromboembolism model.

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Thrombin activatable fibrinolysis inhibitor and its fibrinolytic effect in normal pregnancy

Thrombosis and Haemostasis ◽

10.1160/th04-06-0387 ◽

2004 ◽

Vol 92 (11) ◽

pp. 1025-1031 ◽

Cited By ~ 48

Author(s):

Hatem Mousa ◽

Colin Downey ◽

Zarko Alfirevic ◽

Cheng-Hock Toh

Keyword(s):

Protein S ◽

Significant Negative Correlation ◽

Normal Pregnancy ◽

Clot Lysis ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Significant Drop ◽

Soluble Thrombomodulin ◽

Lysis Time ◽

Fibrinolysis Inhibitor

SummaryWe investigated changes in both thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels and its functional effect on in vitro fibrinolysis in normal pregnancy. 152 pregnant women and 31 women in the immediate postpartum period were studied, with pregnancy divided into 6 windows at 4 weekly intervals. As TAFI influences and is in turn influenced by components of the protein C (PC) pathway, its measurements were correlated with levels of soluble thrombomodulin, PC, protein S (PS) and the overall phenotype of activated PC resistance (APCR). Compared with mean TAFI levels at booking gestation (6.6 +1.2 μg/ml), levels peaked at 35-39 weeks gestation (9.6 +2 μg/ml, p = 0.001), followed by a significant drop within 24 hours of delivery (7.2 + 1.1 μg/ml). In functional terms, the mean clot lysis time (CLT) (101 + 13 min at booking) also peaked at 35-39 weeks gestation (141 + 42 min, p = 0.007) and dropped after delivery (99 + 33 min), and was significantly correlated with gestational age (r = 0.410, p = 0.001) and could be abrogated in the presence of an inhibitor to TAFI activation. A significant negative correlation was found between TAFI levels and APCR (r = −0.478, p <0.001), APCRV (r = −0.598; p <0.001), PS (r = −0.490, P <0.001) and PC (r = −0.198, p = 0.02). In summary, there is a significant increase in TAFI levels, which translates into increased CLT during pregnancy. Furthermore, changes in TAFI contribute to the increasing APCR of pregnancy.

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In Vitro Simulation of Thrombolysis Inhibition

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029607308037 ◽

2008 ◽

Vol 14 (2) ◽

pp. 234-237

Author(s):

Thomas W. Stief

Keyword(s):

Tranexamic Acid ◽

Plasminogen Activator ◽

Microtiter Plate ◽

Caproic Acid ◽

Tissue Type ◽

Clot Lysis ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

In Vitro Simulation

Hyperfibrinolysis is a serious clinical complication. The inhibitory concentrations 50% of antifibrinolytics were analyzed in the microtiter plate clot lysis assay, using 50 µL of plasma clots, 10 µL of antifibrinolytic drug, 10 µL of 354 IU/mL (final) urokinase, 4.46 µg/mL (final) tissue-type plasminogen activator or 0.6 mg/mL plasmin, and 50 µL of pooled normal plasma as clot supernatant. The inhibitory concentrations 50% of lysine against urokinase or tissue-type plasminogen activator is 2.0 or 4.2 mM, against ε-amino-caproic acid 0.7 or 1.5 mM, against tranexamic acid 0.03 or 0.17 mM, respectively. The inhibitory concentrations 50% of lysine, ε-amino-caproic acid, or tranexamic acid against plasmin is 7.4, 0.4, or 0.04 mM. The inhibitory concentrations 50% of aprotinin against urokinase or tissue-type plasminogen activator is about 60 KIU/mL, against plasmin 19 KIU/mL. Lysine might be a new antifibrinolytic drug with a clinically interesting rapid pharmacokinetic. This data help correct dosing of antifibrinolytics to patients with hyperfibrinolysis.

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Fiirst-2: Prospective, Randomized Study Comparing Administration of Clotting Factor Concentrates with Standard Massive Hemorrhage Protocol in Severely Bleeding Trauma Patients

Blood ◽

10.1182/blood-2020-141329 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-6

Author(s):

Luis Da Luz ◽

Jeannie L Callum ◽

Andrew Beckett ◽

Henry T Peng ◽

Paul Engels ◽

...

Keyword(s):

Adverse Events ◽

Interim Analysis ◽

Research Funding ◽

Standard Of Care ◽

Massive Hemorrhage ◽

Clotting Factor ◽

Trauma Patients ◽

Fibrinogen Concentrate ◽

Services Research ◽

The Impact

Introduction: Acute trauma coagulopathy (ATC) is associated with severe hemorrhage. ATC etiology is often multifactorial, with acquired fibrinogen deficiency and consumption of clotting factors recognized as major factors. The FiiRST-2 study will determine the impact of early co-administration of fibrinogen concentrate (FC) and prothrombin complex concentrate (PCC) on the total number of allogeneic blood products (ABPs) transfused compared to the current standard of care (ratio-based plasma resuscitation). Methods: FiiRST-2 is a multicenter (8 Canadian centers), pragmatic, randomized, parallel-control, superiority trial with a two-armed, two-stage design with an adaptive interim analysis. Research ethics board approval has been obtained and the study complies with the Declaration of Helsinki. Trauma patients >16 years old at risk of massive hemorrhage will be randomized to receive FC + PCC or standard of care (ratio-based plasma resuscitation + FC in response to low fibrinogen levels) (Table 1) until all units in the second massive hemorrhage protocol (MHP) pack have been administered, or earlier if the MHP is terminated. The primary endpoint is to demonstrate superiority with respect to the number of ABP units (RBCs [red blood cells] + plasma + platelets) transfused within 24 hours of arrival at the trauma bay/emergency department. Secondary endpoints include RBC units transfused within 24 hours, incidence of thromboembolic events, and ventilator-free days up to Day 28. Safety endpoints include all adverse events (AEs) and serious adverse events (SAEs) up to Day 28. It is projected to enroll 360 patients depending on the results of the Interim analysis. Results: An interim analysis will be performed after 120 patients have completed the study. The study is expected to start late 2020 and enrollment is expected to require about 2 years. Conclusions: FiiRST-2 will determine if early hemostatic therapy with FC + PCC is superior to standard MHP packs in bleeding trauma patients. Results could have a major impact on clinical practice and improve management and outcomes in this high-risk group of patients. Disclosures Da Luz: Octapharma Canada: Research Funding. Callum:Octapharma: Research Funding; Canadian Blood Services: Research Funding. Schwartz:Octapharma: Current Employment. Karkouti:Canadian blood services: Research Funding; Octapharma: Research Funding. OffLabel Disclosure: PCC and fibrinogen concentrate for acquired coagulopathic bleeding (off-label in the USA)

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Differential Inhibition with Antifibrinolytic Agents of Staphylokinase and Streptokinase Induced Clot Lysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653879 ◽

1995 ◽

Vol 73 (05) ◽

pp. 845-849 ◽

Cited By ~ 9

Author(s):

H Roger Lijnen ◽

Jean-Marie Stassen ◽

Désiré Collen

Keyword(s):

Pulmonary Embolism ◽

Tranexamic Acid ◽

Human Plasma ◽

Bolus Injection ◽

Clot Lysis ◽

Antifibrinolytic Agents ◽

Dependent Inhibition ◽

Dose Dependent Inhibition ◽

Dose Dependent

SummaryThe inhibitory effects of antifibrinolytic amino acids on clot lysis induced with recombinant staphylokinase (SakSTAR) or with streptokinase (SK) were evaluated in a human plasma milieu in vitro and in a hamster pulmonary embolism model in vivo. Addition of tranexamic acid to a system composed of 60 μ1 125I-fibrin-labeled plasma clots submerged in 0.5 ml human plasma, caused dose-dependent inhibition of lysis; complete lysis in 120 min required 30 nM SakSTAR or 100 nM SK and was reduced to 50% with 0.015 mM or with 0.07 mM tranexamic acid, respectively. Aprotinin also produced dose-dependent inhibition; lysis with SakSTAR or with SK was reduced to 50% of the control value with 8 KIU/ml or with 10 KIU/ml aprotinin, respectively. Thus, in human plasma in vitro the antifibrinolytic potency of tranexamic acid was 5-fold higher towards SakSTAR than towards SK, whereas that of aprotinin was comparable towards both agents.In hamsters with pulmonary embolism given 0.063 mg/kg SakSTAR or 0.20 mg/kg SK over 30 min, the antifibrinolytic potency of tranexamic acid, administered as a single bolus injection or as a bolus injection followed by continuous infusion, was 8- to 10-fold higher towards SakSTAR than towards SK (50% reduction of clot lysis with SakSTAR at 12.5 mg/kg, as compared to 100-150 mg/kg with SK). In contrast, aprotinin was equipotent towards SakSTAR and SK (50% reduction of clot lysis with 2,000 to 2,700 KlU/kg).The higher antifibrinolytic potency of tranexamic acid (which prevents binding of plasminogen to fibrin) towards SakSTAR than towards SK is most likely related to the requirement of fibrin-bound plasminogen for efficient fibrinolysis with SakSTAR. Tranexamic acid thus may be a useful and relatively specific antidote to clot lysis with SakSTAR and, at higher concentrations, to clot lysis with SK.

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Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Does not Inhibit In Vitro Thrombolysis by Pharmacological Concentrations of t-PA

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615650 ◽

2001 ◽

Vol 85 (04) ◽

pp. 661-666 ◽

Cited By ~ 14

Author(s):

Anna D’Aprile ◽

Alessandra Italia ◽

Paolo Gresele ◽

John Morser ◽

Nicola Semeraro ◽

...

Keyword(s):

Specific Inhibitor ◽

Clot Lysis ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Experimental Conditions ◽

Soluble Thrombomodulin ◽

Blood Clots ◽

Fibrinolysis Inhibitor ◽

Vitro Model

SummaryTAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has been suggested to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has not been properly investigated. We used an in vitro model consisting of 125I-fibrin blood clots submerged in auto-logous defibrinated plasma. Upon addition of t-PA (125-5000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37° C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the generated TAFIa with the specific inhibitor PTI (50 g/ml) or by enhancing TAFI activation through the addition of recombinant soluble thrombomodulin (solulin, 1 μg/ml). In our clot lysis model, activation of TAFI amounted to about 20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant increase in the extent of lysis only at concentrations of t-PA equal to or lower than 250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in those containing pharmacological concentrations of the activator (1000 and 5000 ng/ml). Additional experiments suggest that loss of fibrin-dependence by elevated concentrations of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data indicate that, under our experimental conditions, clot lysis by pharmacological concentrations of t-PA is not influenced by TAFIa even after maximal activation of this procarboxypeptidase.

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A novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) – Part I: Pharmacological characterization

Thrombosis and Haemostasis ◽

10.1160/th06-09-0551 ◽

2007 ◽

Vol 97 (01) ◽

pp. 45-53 ◽

Cited By ~ 16

Author(s):

Lei Zhao ◽

Mariko Nagashima ◽

Jon Vincelette ◽

Drew Sukovich ◽

Weiwei Li ◽

...

Keyword(s):

Peak Plasma ◽

Mutagenic Activity ◽

Plasma Concentrations ◽

Clot Lysis ◽

Significant Activity ◽

Thrombin Activatable Fibrinolysis Inhibitor ◽

Pharmacological Characterization ◽

Drug Concentrations ◽

Fibrinolysis Inhibitor

SummaryWe have discovered a novel small-molecule (3-phosphinoylpropionic acid) inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa), BX 528, which had an IC50 of 2 nM in an enzymatic assay and 50 nM in an in-vitro clot lysis assay, with 3,500- to 35,000-fold selectivity against other carboxypeptidases, such as CPN, CPZ and CPD, and 5- and 12-fold selectivity against CPE (CPH) and CPB, respectively. At 10 µM, BX 528 had no significant activity (< 50% inhibition or antagonism) in a panel of 137 enzymes and receptors. It had no effects on blood coagulation and platelet aggregation up to 300 and 10 µM, respectively. The plasma half-life following intravenous administration was 0.85 hours in rats and 4.5 hours in dogs. No significant metabolism was detected in human, dog or rabbit hepatic microsomes, and no significant inhibition of cytochrome P450 3A4 and 2D6 up to 30 µM. No cytotoxic or cell proliferative effects were found in three hepatic and renal cell lines up to 300 µM and no mutagenic activity was seen in the Ames II screen. There were no significant hemodynamic effects in rats and dogs up to 100 and 30 mg/kg with peak plasma drug concentrations of ~1,000 and 300 µM, respectively. In an in-vivo complement activation model in guinea pigs, BX 528 showed minimal inhibition of plasma CPN activity up to 60 mg/kg with peak plasma concentrations up to 250 µM. Thus, these data demonstrate that BX 528 is a novel, potent, selective and safe TAFIa inhibitor.

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