Identification of heparin interaction sites on thrombin-activatable fibrinolysis inhibitor that modulate plasmin-mediated activation, thermal stability, and antifibrinolytic potential.

Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that provides a molecular link between coagulation and fibrinolysis. Studies have shown that the presence of glycosaminoglycans accelerates TAFI activation by plasmin and stabilizes activated TAFI (TAFIa). Objectives: We aimed to define the elements of TAFI structure that allow these effects. Methods: Based on crystallographic studies and homology to heparin-binding proteins, we performed mutagenesis of surface-exposed charged residues on TAFI that putatively constitute heparin-binding sites. We determined heparin binding, kinetics of activation by plasmin in the presence or absence of heparin, thermal stability, and antifibrinolytic potential of each variant. Results: Mutagenesis of Lys211 and Lys212 did not impair heparin binding but affected the ability of TAFI to be activated by plasmin. Mutagenesis of Lys306 and His308 did not impair heparin binding, but mutation of His308 had a severe negative effect on TAFI/TAFIa function. Mutation of Arg320 and Lys324 in combination markedly decreased heparin binding but had no effect on heparin-mediated acceleration of TAFI activation by plasmin while somewhat decreasing TAFIa stabilization by heparin. Mutagenesis of Lys327 and Arg330 decreased (but did not eliminate) heparin binding while decreasing the ability of heparin to accelerate plasmin-mediated TAFI activation, stabilize TAFIa, and increase the antifibrinolytic ability of TAFIa. A quadruple mutant of Arg320, Lys324, Lys327, and Arg330 completely lost heparin-binding ability and stabilization of the enzyme by heparin. Conclusion: Basic residues in the dynamic flap of TAFIa define a functionally relevant heparin-binding site, but additional heparin-binding sites may be present on TAFI.

Dual antithrombotic therapy dose-dependently alters hemostatic plug structure and function.

BACKGROUND: Antithrombotic medications carry an inherent risk of bleeding, which may be exacerbated when anticoagulant and antiplatelet therapeutics are combined. Prior studies have shown different effects of antiplatelet vs anticoagulant drugs on the structure and function of hemostatic plugs in vivo. OBJECTIVES: We examined whether dual antithrombotic treatment consisting of combined antiplatelet and anticoagulant therapeutics alters hemostatic plug structure and function differently from treatment with either therapeutic alone. METHODS: Mice were treated with the P2Y12 antagonist clopidogrel and the factor Xa inhibitor rivaroxaban across a range of doses, either alone or in combination. The hemostatic response was assessed using a mouse jugular vein puncture injury model. Platelet accumulation and fibrin deposition were evaluated using quantitative multiphoton fluorescence microscopy, and bleeding times were recorded. RESULTS: Mice treated with clopidogrel alone exhibited a decrease in platelet accumulation at the site of injury, with prolonged bleeding times only at the highest doses of clopidogrel used. Mice treated with rivaroxaban alone instead showed a reduction in fibrin deposition with no impact on bleeding. Mice treated with both clopidogrel and rivaroxaban exhibited platelet and fibrin accumulation that was similar to that with either drug given alone; however, dual antithrombotic therapy resulted in impaired hemostasis at doses that had no impact on bleeding when given in isolation. CONCLUSION: Combined administration of antiplatelet and anticoagulant therapeutics exacerbates bleeding as compared to that with either drug alone, potentially via combined loss of both adenosine 5'-diphosphate- and thrombin-mediated platelet activation. These findings enhance our understanding of the bleeding risk associated with dual antithrombotic therapy.

Thrombin spatial distribution determines protein C activation during hemostasis and thrombosis.

Rebalancing the hemostatic system by targeting endogenous anticoagulant pathways, like the protein C (PC) system, is being tested as a means of improving hemostasis in patients with hemophilia. Recent intravital studies of hemostasis demonstrated that, in some vascular contexts, thrombin activity is sequestered in the extravascular compartment. These findings raise important questions about the context-dependent contribution of activated PC (APC) to the hemostatic response, because PC activation occurs on the surface of endothelial cells. We used a combination of pharmacologic, genetic, imaging, and computational approaches to examine the relationships among thrombin spatial distribution, PC activation, and APC anticoagulant function. We found that inhibition of APC activity, in mice either harboring the factor V Leiden mutation or infused with an APC-blocking antibody, significantly enhanced fibrin formation and platelet activation in a microvascular injury model, consistent with the role of APC as an anticoagulant. In contrast, inhibition of APC activity had no effect on hemostasis after penetrating injury of the mouse jugular vein. Computational studies showed that differences in blood velocity, injury size, and vessel geometry determine the localization of thrombin generation and, consequently, the extent of PC activation. Computational predictions were tested in vivo and showed that when thrombin generation occurred intravascularly, without penetration of the vessel wall, inhibition of APC significantly increased fibrin formation in the jugular vein. Together, these studies show the importance of thrombin spatial distribution in determining PC activation during hemostasis and thrombosis.

The contribution of TFPIα to the hemostatic response to injury in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets. OBJECTIVE: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response. METHODS: TFPIα-mutant (TfpiΔα/Δα ) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies. RESULTS: TfpiΔα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the TfpiΔα/Δα mice, no TFPI protein or activity was present in their platelets and thrombin-antithrombin complex levels were indistinguishable from Tfpi+/+ littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the TfpiΔα/Δα mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro- or macrovasculature. CONCLUSION: Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.

Potent reduction of plasma lipoprotein (a) with an antisense oligonucleotide in human subjects does not affect ex vivo fibrinolysis.

It is postulated that lipoprotein (a) [Lp(a)] inhibits fibrinolysis, but this hypothesis has not been tested in humans due to the lack of specific Lp(a) lowering agents. Patients with elevated Lp(a) were randomized to antisense oligonucleotide [IONIS-APO(a)Rx] directed to apo(a) (n = 7) or placebo (n = 10). Ex vivo plasma lysis times and antigen concentrations of plasminogen, factor XI, plasminogen activator inhibitor 1, thrombin activatable fibrinolysis inhibitor, and fibrinogen at baseline, day 85/92/99 (peak drug effect), and day 190 (3 months off drug) were measured. The mean ± SD baseline Lp(a) levels were 477.3 ± 55.9 nmol/l in IONIS-APO(a)Rx and 362.1 ± 89.9 nmol/l in placebo. The mean± SD percentage change in Lp(a) for IONIS-APO(a)Rx was -69.3 ± 12.2% versus -5.4 ± 6.9% placebo (P < 0.0010) at day 85/92/99 and -15.6 ± 8.9% versus 3.2 ± 12.2% (P = 0.003) at day 190. Clot lysis times and coagulation/fibrinolysis-related biomarkers showed no significant differences between IONIS-APO(a)Rx and placebo at all time points. Clot lysis times were not affected by exogenously added Lp(a) at concentrations up to 200 nmol/l to plasma with very low (12.5 nmol/l) Lp(a) levels, whereas recombinant apo(a) had a potent antifibrinolytic effect. In conclusion, potent reductions of Lp(a) in patients with highly elevated Lp(a) levels do not affect ex vivo measures of fibrinolysis; the relevance of any putative antifibrinolytic effects of Lp(a) in vivo needs further study.