Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy

Thrombomodulin (TM) is a type-1 transmembrane glycoprotein encoded by THBD and expressed at high levels on vascular endothelial cells.¹  TM binds thrombin with high affinity and changes its substrate specificity to favor protein C, which when activated to activated protein C (APC) downregulates further thrombin generation by inactivating factors Va and VIIIa.^2,3  Consistent with this downregulatory role, reduced endothelial TM expression has been associated with disseminated intravascular coagulation⁴  and venous thrombosis.⁵  TM has also been linked to the pathogenesis of a dominant bleeding disorder caused by a THBD variant predicting a p.Cys537Stop substitution in the TM transmembrane domain. This results in high plasma TM levels, consistent with shedding of TM extracellular domain from the endothelium.^6,7  It has been proposed that abnormal bleeding in this disorder, hereafter termed TM-associated coagulopathy (TM-AC), results from enhanced TM-mediated APC generation and suppression of further thrombin generation.^6,7 

In addition to mediating APC generation, TM in complex with thrombin is also a potent activator of thrombin-activatable fibrinolysis inhibitor (TAFI).⁸  Activated TAFI (TAFIa) cleaves carboxyl-terminal lysine residues from the fibrin surface thereby reducing binding of plasminogen and tissue plasminogen activator (tPA) and downregulating fibrinolysis.^9,10  Here we describe the impact of high plasma TM levels on TAFI-mediated clot lysis in an unreported pedigree with TM-AC and reveal a previously unrecognized fibrinolytic component of the TM-AC phenotype.

The study cases were enrolled to the National Institute for Health Research (NIHR) BioResources – Rare Diseases (UK REC 13/EE/0325) after informed written consent, in accordance with the Declaration of Helsinki. Plasma TM concentration was measured by ELISA (Abcam, Cambridge, United Kingdom). Calibrated automated thrombography was performed on platelet poor plasma with the PPP LOW reagent (1 pM tissue factor and a Fluoroscan fluorimeter (Thermo-Fisher, Basingstoke, United Kingdom).¹¹  Experiments were also performed in high-TM model blood or plasma using samples from healthy controls spiked with 250 to 2500 ng/mL recombinant C-terminal truncated TM (Peprotech, Hamburg, Germany). THBD was analyzed by whole genome sequencing through the BRIDGE–bleeding and platelet disorders project¹²  and the ThromboGenomics high-throughput platform.¹³ 

Clots were formed in recalcified, diluted plasma with 0.1 U/mL thrombin (Sigma-Aldrich, Poole, United Kingdom) as previously reported¹⁴  in the presence of 300 pM recombinant tPA (Genentech, San Francisco, CA) and 16 μM phospholipids (Rossix, Molndal, Sweden). Clots were formed in the absence or presence of 500 ng/mL TM, 25 μg/mL potato tuber carboxypeptidase inhibitor ([PTCI] a TAFIa inhibitor; Sigma-Aldrich) or 65 μg/mL MA-T12D11 (a monoclonal antibody inhibitor of thrombin/TM-mediated TAFI activation¹⁵ ). Turbidity was monitored every minute at 405 nm.

Clots were formed in recalcified whole blood samples containing 210 pM tPA (Genentech) using the EXTEM reagent and monitored by rotational thromboelastometry (ROTEM; Tem International GmbH, Munchen, Germany), in the presence or absence of 1 to 10 μg/mL PTCI, 25 to 100 μg/mL recombinant activated FVII (rFVIIa; NovoNordisk, Bagsværd, Denmark) or 0.25 to 0.75 U/mL activated prothrombin complex concentrate (aPCC; Baxalta, Bannockburn, IL). Data are expressed as means ± standard error of the mean (SEM) and were analyzed by 2-way analysis of variance with Dunnett’s post hoc test.

The study cases were males aged 59 and 34 years (I.1 and II.2; Figure 1A) with a lifelong propensity for muscle and joint bleeding after minor trauma. Treatment of bleeding with plasma or factor IX infusions was ineffective. However, single 90 μg/kg infusions of rFVIIa (NovoNordisk) were usually sufficient to resolve bleeding. Analysis of plasma by calibrated automated thrombography showed that compared with healthy controls, the cases had reduced endogenous thrombin potential (ETP; 316.3 ± 51.5 nM/min [n = 10] vs 1584.9 ± 56.2 nM/min in controls [n = 20]; P < .01) and reduced peak thrombin concentration (71.7 ± 12.5 nM [n = 10] vs 273.7 ± 19.6 nM in controls [n = 20]; P < .01; Figure 1B-C). Cases and controls showed similar lag time and time to peak thrombin. The plasma TM concentration in the cases was 640.7 ± 21.2 ng/mL (n = 4; reference interval 2.9-7.6 ng/mL). No other abnormalities were observed in coagulation factor or anticoagulant protein levels (supplemental Table 1; available on the Blood Web site).

Consistent with this phenotype, both cases had heterozygous c.1611C>A transversions in the major THBD transcript NM_000361.2, which segregated in the pedigree with abnormal bleeding. This predicted p.Cys537Stop in the TM transmembrane domain, an identical variant to the 2 previously reported TM-AC pedigrees (Figure 1A).^6,7  Addition of sTM to control plasma caused dose-dependent reductions in ETP and peak thrombin, which with 500 ng/mL sTM were similar to the cases (Figure 1C-D). This indicated a causal relationship between high plasma TM levels in the TM-AC cases and reduced thrombin generation. Addition of the bypassing agents rFVIIa or aPCC, which enhance thrombin generation in other coagulopathies, increased ETP in high-TM model plasma, similar to that in pooled FVIII-deficient control plasma samples (P < .01 for ETP with 2-100 μg/mL rFVIIa or 0.25-0.75 U/mL aPCC vs ETP with no bypassing agent). Restoration of ETP was less in the TM-AC plasmas (P < .01 for ETP with 50-100 μg/mL rFVIIa or 0.5-0.75 U/mL aPCC vs ETP with no bypassing agent; Figure 1E-F).

Lysis of TM-AC plasma clots by tPA was significantly slower than controls (time to 50% lysis 175.1 ± 3.4 min [n = 6] vs 103.1 ± 3.9 min in controls [n = 3]; P < .01) and was reproduced in high-TM model plasma (time to 50% lysis 164.1 ± 8.6 min [n = 3]; P < .01; Figure 2A,D). Addition of MA-T12D11, which specifically inhibits thrombin/TM-mediated TAFI activation,¹⁵  completely abrogated delayed fibrinolysis in the TM-AC and high-TM model plasma samples (Figure 2B,D). Inclusion of the TAFIa inhibitor PTCI (25 μg/mL) also reduced the delay in fibrinolysis in TM-AC plasma, although did not completely reduce the 50% lysis time in the high-TM model to control levels (Figure 2C-D).

Clots formed in TM-AC and high-TM model blood samples showed the same maximum clot firmness as controls (Figure 2E; supplemental Table 1), indicating no differences in initial clot viscoelastic strength. However, compared with controls, the time to 90% lysis was significantly delayed in TM-AC blood (time to 90% lysis 56.5 ± 2.3 min [n = 7] vs 29.5 ± 3.5 min in controls [n = 3]; P < .01) and in high-TM model blood (50.4 ± 2.0 [n = 3]; P < .01; Figure 2E-F), indicating delayed fibrinolysis. Addition of 1 to 10 μg/mL PTCI caused a dose-dependent reduction in the delay in fibrinolysis in TM-AC and high-TM model blood samples (Figure 2E-F), reproducing the effects in plasma. Addition of 25 to 100 ng/mL rFVIIa or 0.25 to 0.75 U/mL aPCC, which partially restored thrombin generation in TM-AC and high-TM model samples, did not alter the delayed fibrinolysis in high TM-model blood (Figure 2G).

We confirm that TM-AC is associated with reduced thrombin generation and reveal that delayed fibrinolysis is a component of the TM-AC phenotype using 2 experimental models. We show further that both phenotypes are a consequence of high plasma TM levels and that the delay in fibrinolysis is TAFIa mediated, in line with the established role of TM/thrombin in TAFI activation.^9,10  It is significant that in the presence of high TM levels, enhanced TAFI activation occurred despite reduced thrombin generation and that partial restoration of thrombin generation with rFVIIa or aPCC did not alter the delayed fibrinolysis. This suggests that in TM-AC, TM rather than thrombin is the main determinant of TAFI activation. Our findings highlight the multifactorial role of TM in the regulation of hemostasis and have important implications for the treatment of bleeding in TM-AC.

The authors thank Paul Declerck and Ann Gils, University Leuven, Belgium, for the kind gift of the MA-T12D11 antibody to TAFI.

The NIHR BioResource – Rare Diseases and the ThromboGenomics sequencing projects are supported by the NIHR (http://www.nihr.ac.uk). K.B. is an NIHR academic clinical fellow. S.K.W. is supported by a Medical Research Council (MRC) Clinical Training Fellowship (MR/K023489/1). K.E.S. and E.T. are supported by the NIHR BioResource – Rare Diseases. C.S.W. and N.J.M. are supported by the British Heart Foundation (FS/11/2/28579). A.D.M. is supported by the NIHR Bristol Cardiovascular Biomedical Research Unit.

Contribution: K.B. performed experiments and cowrote the manuscript; C.S.W., M.W., and C.R.-S. performed experiments; S.K.W. and O.G.C. enrolled the study cases and provided the phenotype descriptions; K.E.S. and E.T. designed and performed genetic analyses; N.J.M. designed experiments and cowrote the manuscript; and A.D.M. oversaw the study, designed experiments, and cowrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

A complete list of the members of NIHR BioResource appears in the online appendix.

Correspondence: Andrew D. Mumford, School of Clinical Sciences, University of Bristol, Level 7 Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom; e-mail: a.mumford@bristol.ac.uk.