Lipid specificity of the membrane binding domain of coagulation factor X.

Essentials Membrane-binding GLA domains of coagulation factors are essential for proper clot formation. Factor X (FX) is specific to phosphatidylserine (PS) lipids through unknown atomic-level interactions. Molecular dynamics simulations were used to develop the first membrane-bound model of FX-GLA. PS binding modes of FX-GLA were described, and potential PS-specific binding sites identified. SUMMARY: Background Factor X (FX) binds to cell membranes in a highly phospholipid-dependent manner and, in complex with tissue factor and factor VIIa (FVIIa), initiates the clotting cascade. Experimental information concerning the membrane-bound structure of FX with atomic resolution has remained elusive because of the fluid nature of cellular membranes. FX is known to bind preferentially to phosphatidylserine (PS). Objectives To develop the first membrane-bound model of the FX-GLA domain to PS at atomic level, and to identify PS-specific binding sites of the FX-GLA domain. Methods Molecular dynamics (MD) simulations were performed to develop an atomic-level model for the FX-GLA domain bound to PS bilayers. We utilized a membrane representation with enhanced lipid mobility, termed the highly mobile membrane mimetic (HMMM), permitting spontaneous membrane binding and insertion by FX-GLA in multiple 100-ns simulations. In 14 independent simulations, FX-GLA bound spontaneously to the membrane. The resulting membrane-bound models were converted from HMMM to conventional membrane and simulated for an additional 100 ns. Results The final membrane-bound FX-GLA model allowed for detailed characterization of the orientation, insertion depth and lipid interactions of the domain, providing insight into the molecular basis of its PS specificity. All binding simulations converged to the same configuration despite differing initial orientations. Conclusions Analysis of interactions between residues in FX-GLA and lipid-charged groups allowed for potential PS-specific binding sites to be identified. This new structural and dynamic information provides an additional step towards a full understanding of the role of atomic-level lipid-protein interactions in regulating the critical and complex clotting cascade.

Polyphosphate as modulator of hemostasis, thrombosis, and inflammation.

Inorganic polyphosphate (polyP), a linear polymer of phosphates, is present in many infectious microorganisms and is secreted by mast cells and platelets. PolyP has recently been shown to accelerate blood clotting and slow fibrinolysis, in a manner that is highly dependent on polymer length. Very long-chain polyP (of the type present in microorganisms) is an especially potent trigger of the contact pathway, enhances the proinflammatory activity of histones, and may participate in host responses to pathogens. PolyP also inhibits complement, providing another link between polyP and inflammation/innate immunity. Platelet-size polyP (which is considerably shorter) accelerates factor V activation, opposes the anticoagulant action of tissue factor pathway inhibitor, modulates fibrin clot structure, and promotes factor XI activation. PolyP may have utility in treating bleeding. It is also a potential target for the development of antithrombotic drugs with a novel mechanism of action and potentially fewer bleeding side effects compared with conventional anticoagulants.

Polyphosphate, platelets, and coagulation.

While we have understood the basic outline of the enzymes and reactions that make up the traditional blood coagulation cascade for many years, recently our appreciation of the complexity of these interactions has greatly increased. This has resulted in unofficial 'revisions' of the coagulation cascade to include new amplification pathways and connections between the standard coagulation cascade enzymes, as well as the identification of extensive connections between the immune system and the coagulation cascade. The discovery that polyphosphate is stored in platelet dense granules and is secreted during platelet activation has resulted in a recent burst of interest in the role of this ancient molecule in human biology. Here we review the increasingly complex role of platelet polyphosphate in hemostasis, thrombosis, and inflammation that has been uncovered in recent years, as well as novel therapeutics centered on modulating polyphosphate's roles in coagulation and inflammation.

Factor XI anion-binding sites are required for productive interactions with polyphosphate.

BACKGROUND: Conversion of factor XI (FXI) to FXIa is enhanced by polymers of inorganic phosphate (polyP). This process requires FXI to bind to polyP. Each FXIa subunit contains anion-binding sites (ABSs) on the apple 3 (A3) and catalytic domains that are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. AIMS: To determine the importance of FXI ABSs to polyP enhancement of FXI activation. METHODS: Recombinant FXI variants lacking one or both ABSs were tested in polyP-dependent purified protein systems, plasma clotting assays, and a murine thrombosis model. RESULTS: In the presence of polyP, activation rates for FXI lacking either ABS were reduced compared with wild-type FXI, and FXI lacking both sites had an even greater defect. In contrast to heparin, polyP binding to FXIa did not enhance inhibition by antithrombin and did not interfere with FXIa activation of FIX. FXI lacking one or both ABSs does not reconstitute FXI-deficient plasma as well as wild-type FXI when polyP was used to initiate coagulation. In FXI-deficient mice, FXI lacking one or more ABSs was inferior to wild-type FXI in supporting arterial thrombus formation. CONCLUSIONS: The ABSs on FXIa that are required for expression of heparin's cofactor activity during protease inhibition by antithrombin are also required for expression of polyP cofactor activity during FXI activation. These sites may contribute to FXI-dependent thrombotic processes.

Factor XII promotes blood coagulation independent of factor XI in the presence of long-chain polyphosphates.

BACKGROUND: Inorganic polyphosphates (polyP), which are secreted by activated platelets (short-chain polyP) and accumulate in some bacteria (long-chain polyP), support the contact activation of factor XII (FXII) and accelerate the activation of FXI. OBJECTIVES: The aim of the present study was to evaluate the role of FXI in polyP-mediated coagulation activation and experimental thrombus formation. METHODS AND RESULTS: Pretreatment of plasma with antibodies that selectively inhibit FXI activation by activated FXII (FXIIa) or FIX) activation by activated FXI (FXIa) were not able to inhibit the procoagulant effect of long or short-chain polyP in plasma. In contrast, the FXIIa inhibitor, corn trypsin inhibitor, blocked the procoagulant effect of long and short polyP in plasma. In a purified system, long polyP significantly enhanced the rate of FXII and prekallikrein activation and the activation of FXI by thrombin but not by FXIIa. In FXI-deficient plasma, long polyP promoted clotting of plasma in an FIX-dependent manner. In a purified system, the activation of FXII and prekallikrein by long polyP promoted FIX activation and prothombin activation. In an ex vivo model of occlusive thrombus formation, inhibition of FXIIa with corn trypsin inhibitor but not of FXI with a neutralizing antibodies abolished the prothrombotic effect of long polyP. CONCLUSIONS: We propose that long polyP promotes FXII-mediated blood coagulation bypassing FXI. Accordingly, some polyp-containing pathogens may have evolved strategies to exploit polyP-initiated FXII activation for virulence, and selective inhibition of FXII may improve the host response to pathogens.

Immobilized transition metal ions stimulate contact activation and drive factor XII-mediated coagulation.

BACKGROUND: Upon contact with an appropriate surface, factor XII (FXII) undergoes autoactivation or cleavage by kallikrein. Zn(2+) is known to facilitate binding of FXII and the cofactor, high molecular weight kininogen (HK), to anionic surfaces. OBJECTIVES: To investigate whether transition metal ions immobilized on liposome surfaces can initiate coagulation via the contact pathway. METHODS AND RESULTS: Liposomes containing a metal ion-chelating lipid, 1,2-dioleoyl-sn-glycero-3-{(N[5-amino-1-carboxypentyl]iminodiacetic acid)succinyl} ammonium salt (DOGS-NTA), were prepared by membrane extrusion (20% DOGS-NTA, 40% phosphatidylcholine, 10% phosphatidylserine, and 30% phosphatidylethanolamine). Ni(2+) immobilized on such liposomes accelerated clotting in normal plasma, but not factor XI (FXI)-deficient or FXII-deficient plasma. The results were similar to those obtained with a commercial activated partial thromboplastin time reagent. Charging such liposomes with other transition metal ions revealed differences in their procoagulant capacity, with Ni(2+) > Cu(2+) > Co(2+) and Zn(2+). Plasma could be depleted of FXI, FXII and HK by adsorption with Ni(2+) -containing beads, resulting in longer clot times. Consistent with this, FXI, FXII and HK bound to immobilized Ni(2+) or Cu(2+) with high affinity as determined by surface plasmon resonance. In the presence of Ni(2+) -bearing liposomes, K(m) and k(cat) values derived for autoactivation of FXII and prekallikrein, as well as for activation of FXII by kallikrein or prekallikrein by FXIIa, were similar to literature values obtained in the presence of dextran sulfate. CONCLUSIONS: Immobilized Ni(2+) and Cu(2+) bind FXII, FXI and HK with high affinity and stimulate activation of the contact pathway, driving FXII-mediated coagulation. Activation of the contact system by immobilized transition metal ions may have implications during pathogenic infection or in individuals exposed to high levels of pollution.

Nanoscale studies of protein-membrane interactions in blood clotting.

Most of the steps in the blood clotting cascade require clotting proteins to bind to membrane surfaces with exposed phosphatidylserine. In spite of the importance of these protein-membrane interactions, we still lack a detailed understanding of how clotting proteins interact with membranes and how membranes contribute so profoundly to catalysis. Our laboratories are using multidisciplinary approaches to explore, at atomic-resolution, how blood clotting protein complexes assemble and function on membrane surfaces.

Haplotype and genotype effects of the F7 gene on circulating factor VII, coagulation activation markers and incident coronary heart disease in UK men.

BACKGROUND: Evidence for the associations of single nucleotide polymorphisms (SNPs) in the F7 gene and factor (F)VII levels and with risk of coronary heart disease (CHD) is inconsistent. We examined whether F7 tagging SNPs (tSNPs) and haplotypes were associated with FVII levels, coagulation activation markers (CAMs) and CHD risk in two cohorts of UK men. METHODS: Genotypes for eight SNPs and baseline levels of FVIIc, FVIIag and CAMs (including FVIIa) were determined in 2773 healthy men from the Second Northwick Park Heart Study (NPHS-II). A second cohort, Whitehall II study (WH-II, n = 4055), was used for replication analysis of FVIIc levels and CHD risk. RESULTS: In NPHS-II the minor alleles of three SNPs (rs555212, rs762635 and rs510317; haplotype H2) were associated with higher levels of FVIIag, FVIIc and FVIIa, whereas the minor allele for two SNPs (I/D323 and rs6046; haplotype H5) was associated with lower levels. Adjusted for classic risk factors, H2 carriers had a CHD hazard ratio of 1.34 [95% confidence interval (CI): 1.12-1.59; independent of FVIIc], whereas H5 carriers had a CHD risk of 1.29 (95% CI: 1.01-1.56; not independent of FVIIc) and significantly lower CAMs. Effects of haplotypes on FVIIc levels were replicated in WH-II, as was the association of H5 with higher CHD risk [pooled-estimate odds ratio (OR) 1.16 (1.00-1.36), P = 0.05], but surprisingly, H2 exhibited a reduced risk for CHD. CONCLUSION: tSNPs in the F7 gene strongly influence FVII levels. The haplotype associated with low FVIIc level, with particularly reduced functional activity, was consistently associated with increased risk for CHD, whereas the haplotype associated with high FVIIc level was not.

Dynamical view of membrane binding and complex formation of human factor VIIa and tissue factor.

SUMMARY BACKGROUND: The molecular mechanism of enhancement of the enzymatic activity of factor VIIa by tissue factor (TF) is not fully understood, primarily because of the lack of atomic models for the membrane-bound form of the TF-FVIIa complex. OBJECTIVES: To construct the first membrane-bound model of the TF-FVIIa complex, and to investigate the dynamics of the complex in solution and on the surface of anionic membranes by using large-scale molecular dynamics (MD) simulations in full atomic detail. METHODS: Membrane-bound models of the TF-FVIIa complex and the individual factors were constructed and subjected to MD simulations, in order to characterize protein-protein and protein-lipid interactions, and to investigate the dynamics of TF and FVIIa. RESULTS: The MD trajectories reveal that isolated FVIIa undergoes large structural fluctuation, primarily due to the hinge motions between its domains, whereas soluble TF (sTF) is structurally stable. Upon complex formation, sTF restricts the motion of FVIIa significantly. The results also show that, in the membrane-bound form, sTF directly interacts with the lipid headgroups, even in the absence of FVIIa. CONCLUSION: The first atomic models of membrane-bound sTF-FVIIa, FVIIa and sTF are presented, revealing that sTF forms direct contacts with the lipids, both in the isolated form and in complex with FVIIa. The main effect of sTF binding to FVIIa is spatial stabilization of the catalytic site of FVIIa, which ensures optimal interaction with the substrate, FX.

Polyphosphate binds with high affinity to exosite II of thrombin.

BACKGROUND: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. OBJECTIVE: To examine the interaction of polyphosphate with thrombin. METHODS AND RESULTS: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin's C-terminal dodecapeptide and gamma-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na(+)-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (K(d) approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin. CONCLUSION: Polyphosphate interacts with thrombin's exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nM K(d) for the polyphosphate-thrombin interaction.

Protein-membrane interactions: blood clotting on nanoscale bilayers.

The clotting cascade requires the assembly of protease-cofactor complexes on membranes with exposed anionic phospholipids. Despite their importance, protein-membrane interactions in clotting remain relatively poorly understood. Calcium ions are known to induce anionic phospholipids to cluster, and we propose that clotting proteins assemble preferentially on such anionic lipid-rich microdomains. Until recently, there was no way to control the partitioning of clotting proteins into or out of specific membrane microdomains, so experimenters only knew the average contributions of phospholipids to blood clotting. The development of nanoscale membrane bilayers (Nanodiscs) has now allowed us to probe, with nanometer resolution, how local variations in phospholipid composition regulate the activity of key protease-cofactor complexes in blood clotting. Furthermore, exciting new progress in solid-state NMR and large-scale molecular dynamics simulations allow structural insights into interactions between proteins and membrane surfaces with atomic resolution.

Polyphosphate as a general procoagulant agent.

BACKGROUND: Polyphosphate is secreted by activated platelets and we recently showed that it accelerates blood clotting, chiefly by triggering the contact pathway and promoting factor (F) V activation. RESULTS: We now report that polyphosphate significantly shortened the clotting time of plasmas from patients with hemophilia A and B and that its procoagulant effect was additive to that of recombinant FVIIa. Polyphosphate also significantly shortened the clotting time of normal plasmas containing a variety of anticoagulant drugs, including unfractionated heparin, enoxaparin (a low molecular weight heparin), argatroban (a direct thrombin inhibitor) and rivaroxaban (a direct FXa inhibitor). Thromboelastography revealed that polyphosphate normalized the clotting dynamics of whole blood containing these anticoagulants, as indicated by changes in clot time, clot formation time, alpha angle, and maximum clot firmness. Experiments in which preformed FVa was added to plasma support the notion that polyphosphate antagonizes the anticoagulant effect of these drugs via accelerating FV activation. Polyphosphate also shortened the clotting times of plasmas from warfarin patients. CONCLUSION: These results suggest that polyphosphate may have utility in reversing anticoagulation and in treating bleeding episodes in patients with hemophilia.

Relationship between markers of activated coagulation, their correlation with inflammation, and association with coronary heart disease (NPHSII).

OBJECTIVE: To determine whether activation of coagulation increases in parallel with inflammation and whether coagulation activation markers (CAMs) are independently associated with coronary heart disease (CHD), in the prospective study, NPHSII. METHODS: Surveillance of 2997 men between 50 and 63 years yielded 314 first CHD events during 36507 person-years of observation. The plasma levels of activated factor XII (FXIIa), the peptides released upon activation of factor X (FXpep) and factor IX (FIXpep), activated factor VII (FVIIa), prothrombin fragment 1 + 2 (F1 + 2) and fibrinopeptide A (FpA) served as indices of activity along the coagulation pathway. C reactive protein (CRP) provided a marker of inflammatory activity. RESULTS: While borderline or significant correlations were identified for each CAM with inflammation, as determined by CRP levels, these did not reach as high a numerical value as was shown for fibrinogen with CRP. FVIIa and FIXpep possessed independent associations with CHD: a one SD increase in adjusted FIXpep and FVIIa level was associated with a relative hazard of 1.20 (95% CI 1.00-1.43) and 0.70 (CI 0.58-0.86), respectively, using a group including all CHD events, compared with 'no-event'. CONCLUSIONS: Inflammation has significant but minimal impact upon CAMs of the extrinsic coagulation pathway. Reduced FVIIa and increased FIXpep levels were found to be significant, independent, predictors of CHD.

Traces of factor VIIa modulate thromboplastin sensitivity to factors V, VII, X, and prothrombin.

BACKGROUND: Thromboplastin reagents are used to conduct prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and screen for clotting factor deficiencies. Thromboplastins made from purified, recombinant tissue factor are generally more sensitive to changes in plasma factor (F) VII levels than are thromboplastins prepared from tissue extracts. This may be problematic as FVII's short plasma half-life can result in day-to-day fluctuation during oral anticoagulant therapy. We hypothesized that trace contamination of tissue-derived thromboplastins with FVII(a) blunts sensitivity to plasma FVII levels. METHODS: Traces of purified FVIIa were added to thromboplastin reagents prepared using recombinant human tissue factor and the effect on sensitivity to individual clotting factors was quantified in PT clotting assays. RESULTS AND CONCLUSIONS: Adding 5-100 pm FVIIa not only decreased thromboplastin sensitivity to plasma FVII, it surprisingly increased sensitivity to plasma levels of FV, FX and prothrombin. In addition, traces of FVIIa interacted with changes in the salt content and phospholipid composition of recombinant thromboplastins to further modulate their sensitivities to individual clotting factors. These results help explain how thromboplastin reagents of differing composition exhibit differing sensitivities to individual clotting factor levels. Implications of our results for monitoring oral anticoagulant therapy and other uses of the PT assay are discussed.

Phospholipid composition controls thromboplastin sensitivity to individual clotting factors.

BACKGROUND: Tissue factor is the active ingredient in thromboplastin reagents used to perform prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and to screen for clotting factor deficiencies. Thromboplastins are complex mixtures prepared from extracts of brain or placenta, although newer thromboplastins contain recombinant tissue factor incorporated into phospholipid vesicles. Thromboplastins can vary widely in their sensitivity to reductions in the levels of vitamin K-dependent clotting factors. A system to compensate for this, the International Sensitivity Index (ISI) and International Normalized Ratio (INR), has revolutionized the monitoring of oral anticoagulant therapy. The INR system is also sometimes used to monitor coagulopathies in patients with sepsis or liver failure, applications for which it was not originally designed and for which it has not been rigorously validated. OBJECTIVES: To better understand thromboplastin performance, we systematically investigated which properties of recombinant thromboplastins influence their sensitivities to changes in the levels of specific clotting factors. RESULTS: We now report that relative sensitivities to changes in the plasma levels of factors V, VII, X (FV, FVII, FX) and prothrombin are differentially influenced by a recombinant thromboplastin's content of phospholipid and sodium chloride. Furthermore, thromboplastins of similar ISI values may exhibit quite different sensitivities to each of these clotting factors. CONCLUSIONS: Differing sensitivities of thromboplastin reagents to individual clotting factor levels have implications for monitoring of oral anticoagulant therapy and interpreting results of the PT assay.

Properties of recombinant human thromboplastin that determine the International Sensitivity Index (ISI).

Prothrombin Time (PT) clotting tests are widely used to monitor oral anticoagulation therapy and to screen for clotting factor deficiencies. The active ingredient in PT reagents (thromboplastins) is tissue factor, the integral membrane protein that triggers the clotting cascade through the extrinsic pathway. Several years ago, a system for calibrating and using thromboplastin reagents, known as the International Sensitivity Index (ISI) and the International Normalized Ratio (INR), was developed to standardize monitoring of oral anticoagulant therapy. The ISI/INR method, while revolutionizing the monitoring of coumarin therapy, has been criticized for a number of perceived shortcomings. We have undertaken a series of studies aimed at achieving a detailed understanding of which parameters influence the ISI values of thromboplastin reagents, with an ultimate goal of creating 'designer thromboplastins' whose sensitivities to the various clotting factors can be individually tailored. In this study, we demonstrate that ISI values of thromboplastin reagents based on relipidated, recombinant human tissue factor can be controlled by a combination of changes in the phospholipid content (in particular, the levels of phosphatidylserine and phosphatidylethanolamine) and ionic strength. The sensitivity of a given thromboplastin reagent can be increased (i.e. its ISI value decreased) by decreasing the content of phosphatidylserine and/or increasing the ionic strength. The molar ratio of phospholipid to tissue factor, on the other hand, had essentially no impact on ISI value.

Rapid and efficient incorporation of tissue factor into liposomes.

Tissue factor (TF), the physiological trigger of the blood clotting cascade, is also the active ingredient in thromboplastin preparations which are widely used in clotting assays such as the prothrombin time (PT) test. A type I integral membrane protein, TF must be incorporated into suitable phospholipid membranes for full procoagulant activity. Several methods exist for incorporating TF into phospholipid vesicles, typically employing the formation of mixed micelles containing detergent, phospholipid and TF, followed by detergent removal or dilution below the critical micelle concentration (CMC). These methods have certain drawbacks: they may take several days to complete, employ expensive detergents, are difficult to scale up, and do not always result in complete detergent removal. In this study we have investigated the use of a variety of detergents [Triton X-100, octaethylene glycol monododecyl ether (C(12)E(8)), cholate, deoxycholate, and n-octyl-beta-D-glucopyranoside], and the use of adsorbent beads (Bio-Beads SM-2) for removing detergent, in processes to incorporate TF into proteoliposomes with high specific activity in coagulation assays. The method we have developed is rapid and readily scalable, yielding thromboplastin preparations with specific activities in plasma clotting assays that are at least as high as those made with detergent dialysis.