Blood coagulation factor X/Xa sits at a pivotal point in the coagulation cascade and has a role in each of the three major pathways (intrinsic, extrinsic and the common pathway). Due to this central ...position, it is an attractive therapeutic target to either enhance or dampen thrombin generation. In this brief review, I will summarize key developments in the molecular understanding of this critical clotting factor and discuss the molecular basis of FX deficiency, highlight difficulties in expressing recombinant factor X, and detail two factor X variants evaluated clinically.
The membrane-dependent interaction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation essential for hemostasis. Activated platelets are considered to provide ...the primary biological surface to support prothrombinase function. However, the question of how other cell types may cooperate within the biological milieu to affect hemostatic plug formation remains unaddressed. We used confocal fluorescence microscopy to image the distribution of site-specific fluorescent derivatives of FVa and FXa after laser injury in the mouse cremaster arteriole. These proteins bound to the injury site extend beyond the platelet mass to the surrounding endothelium. Although bound FVa and FXa may have been present on the platelet core at the nidus of the injury, bound proteins were not evident on platelets adherent even a small distance from the injury site. Manipulations to drastically reduce adherent platelets yielded a surprisingly modest decrease in bound FXa and FVa with little impact on fibrin formation. Thus, platelets adherent to the site of vascular injury do not play the presumed preeminent role in supporting prothrombinase assembly and thrombin formation. Rather, the damaged/activated endothelium and possibly other blood cells play an unexpectedly important role in providing a procoagulant membrane surface in vivo.
•Prothrombinase bound in the vicinity of vascular damage is distributed away from platelets and is largely found on activated endothelium.•The activated endothelium plays an unexpectedly important role in supporting prothrombinase assembly and function at the site of damage.
Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of ...inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1′ bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.
•The endogenous inhibitors of APC also inhibit other coagulation proteases rendering them unacceptable for treatment of hemophilia.•Rationally designed APC-specific serpins rescue thrombin generation in vitro and restore hemostasis in hemophilia mouse models.
Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the ...proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963–1008 and acidic region 2, AR2; 1493–1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (μM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.
Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need ...in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Blood coagulation factor V (FV) plays a pivotal role in blood coagulation. It is found in both plasma and in platelets and has a profound impact on thrombin generation. Deficiency of this clotting ...factor due to inherited or acquired conditions results in a broad spectrum of bleeding symptoms. Surprisingly however, some patients with undetectable levels of FV experience relatively mild bleeding. The aim of this review is to highlight this rare coagulation factor disorder and touch upon its clinical manifestations, diagnosis, and treatment. Furthermore, recent advances that shed new light on the importance of platelet FV and other modifiers which influence bleeding tendencies in severe FV deficiency will be discussed.
•Endogenous plasma protein anticoagulants limit clot formation by inhibiting FIXa activity.•Delaying FIXa inhibition using zymogen-like variants increases the potency of administered FIX in ...hemophilia B models.
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Factor IXa (FIXa) plays a pivotal role in coagulation by contributing to FX activation via the intrinsic pathway. Although antithrombin (AT) and other plasma inhibitors are thought to regulate FIXa procoagulant function, the impact of FIXa inhibition on thrombin generation and clot formation in vivo remains unclear. Here, we generated FIXa variants with altered reactivity to plasma inhibitors that target the FIXa active site but maintain procoagulant function when bound to its cofactor, FVIIIa. We found that selected FIXa variants (eg, FIXa-V16L) have a prolonged activity half-life in the plasma due, in part, to AT resistance. Studies using hemophilia B mice have shown that delayed FIXa inhibition has a major impact on reducing the bleeding phenotype and promoting thrombus formation following administration of FIX protein. Overall, these results demonstrate that the regulation of FIXa inhibition contributes in a major way to the spatial and temporal control of coagulation at the site of vascular injury. Our findings provide novel insights into the physiological regulation of FIXa, enhance our understanding of thrombus formation in vivo via the intrinsic pathway, and suggest that altering FIXa inhibition could have therapeutic benefits.
Endogenous plasma protein anticoagulants limit clot formation by inhibiting factor IXa activity. Ivanciu and colleagues show that factor-delaying IXa inhibition using novel zymogen-like variants increases the potency of administered factor IX in models of hemophilia B. The authors’ data enhance our understanding of the control of clot formation, and the findings suggest a novel strategy to augment clot formation in hemophilia B.
Activated protein C (APC) is an important anticoagulant protein that regulates thrombin generation through inactivation of factor V (FV) and activated factor V (FVa). The rate of APC inactivation of ...FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg306, Arg506, and Arg679). The molecular basis for FV resistance to APC is unknown. Further, there is no information about how FV-short, a physiologically relevant isoform of FV with a shortened B-domain, is regulated by APC. Here, we identify the molecular determinants which differentially regulate APC recognition of FV versus FVa and uncover how FV-short can be protected from this anticoagulant pathway. Using recombinant FV derivatives and B-domain fragments, we show that the conserved basic region (BR; 963–1008) within the central portion of the B-domain plays a major role in limiting APC cleavage at Arg506. Derivatives of FV lacking the BR, including FV-short, were subject to rapid cleavage at Arg506 and were inactivated like FVa. The addition of a FV-BR fragment reversed this effect and delayed APC inactivation. We also found that anticoagulant glycoprotein TFPIα, which has a C-terminal BR homologous to FV-BR, protects FV-short from APC inactivation by delaying cleavage at Arg506. We conclude that the FV-BR plays a major role in protecting FV from APC inactivation. Using a similar mechanistic strategy, TFPIα also shields FV-short from APC. These findings clarify the resistance of FV to APC, advance our understanding of FV/FVa regulation, and establish a mechanistic framework for manipulating this reaction to alter coagulation.
Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and ...TFPIβ. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV in an inactive conformation via interaction with an acidic region of the B domain. We demonstrate rapid inhibition of prothrombinase by TFPIα mediated through a high-affinity exosite interaction between the basic region of TFPIα and the FV acidic region, which is retained in FXa-activated FVa and platelet FVa. This inhibitory activity is not mediated by TFPIβ and is lost upon removal of the acidic region of FVa by thrombin. The data identify a previously undescribed, isoform-specific anticoagulant function for TFPIα and are a unique description of physiologically relevant inhibition of prothrombinase. These findings, combined with previous descriptions of differential expression patterns of TFPIα and TFPIβ in platelets and endothelial cells, suggest that the TFPI isoforms may act through distinct mechanisms to inhibit the initial stages of intravascular coagulation, with TFPIβ acting to dampen TF expressed on the surface of vascular cells, whereas TFPIα dampens the initial prothrombinase formed on the activated platelet surface.
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