Plasma fibrin clots of pulmonary embolism patients present increased amounts of factor XIII and alpha2-antiplasmin at 3 months' anticoagulation since the acute phase.

Fibrin cross-linking by coagulation factor (F)XIII leads to clot stabilization. Reduced plasma FXIII levels have been reported in acute pulmonary embolism (PE) patients. We investigated the impact of anticoagulant therapy on clot-bound amounts of FXIII and α2-antiplasmin and their associations with fibrin clot properties in patients with PE. Clots generated from plasma of 18 acute symptomatic patients on admission and after a 3-month treatment with rivaroxaban were assessed off anticoagulation using mass spectrometry. Plasma FXIII and α2-antiplasmin activity were determined at the 2 time points along with thrombin generation markers, plasma fibrin clot permeability (Ks), and clot lysis time (CLT). Following anticoagulant therapy, clot-bound FXIII increased from 2.97 (interquartile range, 1.98 - 4.08) to 4.66 (3.5 - 6.9) mg/g protein and α2-antiplasmin from 9.4 (7.2 - 10.6) to 11 (9.5 - 14) mg/g protein (both p < 0.0001). The two parameters showed positive correlation at baseline only (r = 0.63, p = 0.0056). Similarly to clot-bound amounts, plasma FXIII (+25.8%) and α2-antiplasmin activity (+12%) increased at 3 months. Plasma FXIII activity on admission, but not after 3 months since the index PE, was associated with amounts of clot-bound FXIII (r = 0.35, p = 0.043) and α2-antiplasmin (r = 0.47, p = 0.048). At baseline, clot-bound FXIII correlated with plasma F1+2 prothrombin fragments levels (r = 0.51, p = 0.03), while clot-bound α2-antiplasmin correlated with CLT (r = 0.43, p = 0.036). At 3 months associations of clot-bound FXIII and α2-antiplasmin were abolished. This study assessed for the first time changes in the fibrin clot composition following acute PE, suggesting an increase of clot-bound and plasma FXIII and α2-antiplasmin levels after 3 months.

Factor XIII: novel structural and functional aspects.

Factor (F)XIII is a protransglutaminase that, in addition to maintaining hemostasis, has multiple plasmatic and intracellular functions. Its plasmatic form (pFXIII) is a tetramer of two potentially active A (FXIII-A) and two inhibitory/carrier B (FXIII-B) subunits, whereas its cellular form (cFXIII) is a dimer of FXIII-A. FXIII-A belongs to the family of transglutaminases (TGs), which show modest similarity in the primary structure, but a high degree of conservatism in their domain and sub-domain secondary structure. FXIII-A consists of an activation peptide, a ß-sandwich, a catalytic and two ß-barrel domains. FXIII-B is a glycoprotein consisting of 10 repetitive sushi domains each held together by two internal disulfide bonds. The structural elements of FXIII-A involved in the interaction with FXIII-B have not been elucidated; in FXIII-B the first sushi domain seems important for complex formation. In the circulation pFXIII is bound to the fibrinogen γ'-chain through its B subunit. In the process of pFXIII activation first thrombin cleaves off the activation peptide from FXIII-A, then in the presence of Ca(2+) FXIII-B dissociates and FXIII-A becomes transformed into an active transglutaminase (FXIIIa). The activation is highly accelerated by the presence of fibrin(ogen). cFXIII does not require proteolysis for intracellular activation. The three-dimensional structure of FXIIIa has not been resolved. Based on analogies with transglutaminase-2, a three-dimensional structure of FXIIIa was developed by molecular modeling, which shows good agreement with the drastic structural changes demonstrated by biochemical studies. The structural requirements for enzyme-substrate interaction and for transglutaminase activity are also reviewed.

The involvement of blood coagulation factor XIII in fibrinolysis and thrombosis.

It has been known for a long time that blood coagulation factor XIII (FXIII) is essential for maintaining haemostasis, its deficiency leads to severe bleeding complication. Biochemical studies have revealed that FXIII is a key regulator of fibrinolysis and, in addition to its role in haemostasis, it has also been implicated in the pathology of arterial and venous thrombosis. Most recently, the polymorphisms in the FXIII subunit genes and their influence on the risk of thrombotic diseases have stirred a lot of interest. This review, besides including the basic biochemistry of FXIII, mainly concentrates on the biochemical and clinical aspects of the involvement of FXIII in fibrinolysis and thrombosis. Biochemical aspects: Basics on the structure and activation of plasma and cellular FXIII. The enzymological features of activated FXIII and its main substrates. The interaction of FXIIIa with fibrinogen/fibrin and with components of the fibrinolytic system. The impact of cross-linked fibrin clot formation on the fibrinolytic processes. The down-regulation of FXIIIa within the fibrin clot. FXIII polymorphisms and their biochemical consequences. Clinical Aspects: FXIII level and the risk of arterial thrombosis (coronary artery disease, peripheral artery disease, ischemic stroke). The effect of FXIII subunit polymorphisms on the risk of arterial thrombotic diseases. The interplay between FXIII polymorphisms and other factors influencing the risk of arterial thrombosis. FXIII and venous thromboembolism.

4-thio-deoxyuridylate-modified thrombin aptamer and its inhibitory effect on fibrin clot formation, platelet aggregation and thrombus growth on subendothelial matrix.

BACKGROUND: The consensus thrombin aptamer C15-mer is a single-stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4-thio-deoxyuridylate (s4dU)-containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. METHODS: Three different analogs of the original thrombin-inhibiting sequence, in which some of the thymidylate residues were replaced by 4-thio-deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin-catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin-induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin-treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. RESULTS: As compared with the C15-mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15-mer) showed a 2-fold increased inhibition of thrombin-catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin-treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin-induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15-mer was 3-fold and twelve-fold more effective than C15-mer, respectively. CONCLUSION: The replacement of four thymidylate residues in C15-mer by 4-thio-deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.