Generation of an anticoagulant aptamer that targets factor V/Va and disrupts the FVa-membrane interaction in normal and COVID-19 patient samples.

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.

Anticoagulant effects of statins and their clinical implications.

There is evidence indicating that statins (3-hydroxy-methylglutaryl coenzyme A reductase inhibitors) may produce several cholesterol-independent antithrombotic effects. In this review, we provide an update on the current understanding of the interactions between statins and blood coagulation and their potential relevance to the prevention of venous thromboembolism (VTE). Anticoagulant properties of statins reported in experimental and clinical studies involve decreased tissue factor expression resulting in reduced thrombin generation and attenuation of pro-coagulant reactions catalysed by thrombin, such as fibrinogen cleavage, factor V and factor XIII activation, as well as enhanced endothelial thrombomodulin expression, resulting in increased protein C activation and factor Va inactivation. Observational studies and one randomized trial have shown reduced VTE risk in subjects receiving statins, although their findings still generate much controversy and suggest that the most potent statin rosuvastatin exerts the largest effect.

Restoring the procofactor state of factor Va-like variants by complementation with B-domain peptides.

Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism of this FV autoinhibition. We show that a fragment encoding the basic region (BR) of the B-domain binds with high affinity to cofactor-like FV(a) variants that harbor an intact acidic region. Furthermore, the BR inhibits procoagulant function of the variants, thereby restoring the procofactor state. The BR competes with FXa for binding to FV(a), and limited proteolysis of the B-domain, specifically at Arg(1545), ablates BR binding to promote high affinity association between FVa and FXa. These results provide new insight into the mechanism by which the B-domain stabilizes FV as an inactive procofactor and reveal how limited proteolysis of FV progressively destabilizes key regulatory regions of the B-domain to produce an active form of the molecule.

Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.

Novel antithrombotic strategies in cardiovascular diseases.

Ischemic cardiovascular diseases represent the most common cause of mortality and morbidity in the western world, and atherothrombosis occupies a central role in their pathophysiology. Venous thrombi, which form under low shear conditions, are predominantly composed of fibrin and red cells, while arterial thrombi form under high shear conditions and are composed primarily of platelet aggregates held together by fibrin strands. Several successful strategies targeting specific steps in coagulation and platelet function or interaction have been developed to prevent or treat atherothrombotic disorders. Intense research is currently underway in an effort to develop more safe and effective compounds, such that novel antithrombotics are emerging to target specific steps in the coagulation cascade, as well as in pathways of platelet adhesion, activation and aggregation. This review will focus on the recent advances in research in this fast-evolving field.

New anticoagulant therapy.

The development of new anticoagulants is expanding the list of drugs that can be used to prevent and treat venous and arterial thrombosis. New parenteral anticoagulants have been developed to overcome the limitations of heparin and low-molecular-weight heparin, whereas novel orally active anticoagulants have been designed to provide more streamlined therapy than vitamin K antagonists. This review identifies the molecular targets of new anticoagulants, describes the results of clinical trials, and provides clinical perspective on the opportunities for new anticoagulants.

Altered inactivation pathway of factor Va by activated protein C in the presence of heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'306) or after cleavage at Arg506 (k506) and subsequent cleavage at Arg306 (k306). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k506 by UFH was 12-fold, with the secondary cleavage at Arg306 (k306) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'306) two- to threefold. Low molecular weight heparin (Fragmin) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.

Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323-331 of factor V represent an interactive site for activated factor X.

We have recently shown that amino acid region 307-348 of factor Va heavy chain (42 amino acids, N42R) is critical for cofactor activity and may contain a binding site for factor Xa and/or prothrombin [(2001) J. Biol. Chem. 276, 18614-18623]. To ascertain the importance of this region for factor Va cofactor activity, we have synthesized eight overlapping peptides (10 amino acid each) spanning amino acid region 307-351 of the heavy chain of factor Va and tested them for inhibition of prothrombinase activity. The peptides were also tested for the inhibition of the binding of factor Va to membrane-bound active site fluorescent labeled Glu-Gly-Arg human factor Xa ([OG488]-EGR-hXa). Factor Va binds specifically to membrane-bound [OG488]-EGR-hXa (10nM) with half-maximum saturation reached at approximately 6 nM. N42R was also found to interact with [OG488]-EGR-hXa with half-maximal saturation observed at approximately 230 nM peptide. N42R was found to inhibit prothrombinase activity with an IC50 of approximately 250 nM. A nonapeptide containing amino acid region 323-331 of factor Va (AP4') was found to be a potent inhibitor of prothrombinase. Kinetic analyses revealed that AP4' is a noncompetitive inhibitor of prothrombinase with respect to prothrombin, with a K(i) of 5.7 microM. Thus, the peptide interferes with the factor Va-factor Xa interaction. Displacement experiments revealed that the nonapeptide inhibits the direct interaction of factor Va with [OG488]-EGR-hXa (IC50 approximately 7.5 microM). The nonapeptide was also found to bind directly to [OG488]-EGR-hXa and to increase the catalytic efficiency of factor Xa toward prothrombin in the absence of factor Va. In contrast, a peptadecapeptide from N42R encompassing amino acid region 337-351 of factor Va (P15H) had no effect on either prothrombinase activity or the ability of the cofactor to interact with [OG488]-EGR-hXa. Our data demonstrate that amino acid sequence 323-331 of factor Va heavy chain contains a binding site for factor Xa.

Protein S Gla-domain mutations causing impaired Ca(2+)-induced phospholipid binding and severe functional protein S deficiency.

We have identified 2 PROS1 missense mutations in the exon that encodes the vitamin K-dependent Gla domain of protein S (Gly11Asp and Thr37Met) in kindred with phenotypic protein S deficiency and thrombosis. In studies using recombinant proteins, substitution of Gly11Asp did not affect production of protein S but resulted in 15.2-fold reduced protein S activity in a factor Va inactivation assay. Substitution of Thr37Met reduced expression by 33.2% (P <.001) and activity by 3.6-fold. The Gly11Asp variant had 5.4-fold reduced affinity for anionic phospholipid vesicles (P <.0001) and decreased affinity for an antibody specific for the Ca(2+)-dependent conformation of the protein S Gla domain (HPS21). Examination of a molecular model suggested that this could be due to repositioning of Gla29. In contrast, the Thr37Met variant had only a modest 1.5-fold (P <.001), reduced affinities for phospholipid and HPS21. This mutation seems to disrupt the aromatic stack region. The proposita was a compound heterozygote with free protein S antigen levels just below the lower limit of the normal range, and this is now attributed to the partial expression defect of the Thr37Met mutation. The activity levels were strongly reduced to 15% of normal, probably reflecting the functional deficit of both protein S variants. Her son (who was heterozygous only for Thr37Met) had borderline levels of protein S antigen and activity, reflecting the partial secretion and functional defect associated with this mutation. This first characterization of natural protein S Gla-domain variants highlights the importance of the high affinity protein S-phospholipid interaction for its anticoagulant role.

Isolation and characterization of an antifactor V antibody causing activated protein C resistance from a patient with severe thrombotic manifestations.

A 44-year-old woman with a history of severe thrombotic manifestations presented with a markedly reduced activated protein C-sensitivity ratio (APC-SR). DNA sequencing of and around the regions encoding the APC cleavage sites in the factor Va molecule excluded the presence of the factor VLeiden mutation and of other known genetic mutations. No antiphospholipid antibodies were present in the patient's plasma and both prothrombin time and activated partial thromboplastin time were normal. The total immunoglobulin fraction was isolated from the patient's plasma and found to induce severe APC resistance when added to normal plasma and to factor V-deficient plasma supplemented with increasing concentrations of factor V. Immunoblotting and immunoprecipitation experiments with the total immunoglobulin fraction purified from the patient's plasma demonstrated that the antibody recognizes factor V, is polyclonal, and has conformational epitopes on the entire factor V molecule (heavy and light chains, and B region). Thus, the immunoglobulin fraction interferes with the anticoagulant pathway involving factor V. The inhibitor was isolated by sequential affinity chromatography on protein G-Sepharose and factor V-Sepharose. The isolated immunoglobulin fraction inhibited factor Va inactivation by APC because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the cofactor. The isolated immunoglobulin fraction was also found to inhibit the cofactor effect of factor V for the inactivation of factor VIII by the APC/protein S complex. Our data provide for the first time the demonstration of an antifactor V antibody not related to the presence of antiphospholipid antibodies, which is responsible for thrombotic rather than hemorrhagic symptoms.

Bothrojaracin, a proexosite I ligand, inhibits factor Va-accelerated prothrombin activation.

Bothrojaracin (BJC) is a 27 kDa snake venom protein from Bothrops jararaca that has been characterized as a potent ligand (KD = 75 nM) of human prothrombin (Monteiro RQ, Bock PE, Bianconi ML, Zingali RB, Protein Sci 2001; 10: 1897-904). BJC binds to the partially exposed anion-binding exosite I (proexosite I) forming a stable 1:1, non-covalent complex with the zymogen whereas no interaction with fragment 1 or 2 domains is observed. In addition, BJC interacts with thrombin through exosites I and II (KD = 0.7 nM), and influences but does not block the proteinase catalytic site. In the present work we studied the effect of BJC on human prothrombin activation by factor Xa in the absence or in the presence of its cofactors, factor Va and phospholipids. In the absence of phospholipids, BJC strongly inhibited (80%) the zymogen activation by factor Xa in the presence but not in the absence of factor Va, suggesting a specific interference in the cofactor activity. In the presence of phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine), BJC also inhibited (35%) prothrombin activation by factor Xa in the presence but not in the absence of factor Va. BJC showed a higher inhibitory effect (70%) towards thrombin formation by prothrombinase complex assembled on phospholipid vesicles composed by 95% phosphatidylcholine, 5% phosphatidylserine. Activation of prothrombin by platelet-assembled prothrombinase complex (factor Xa, factor Va and thrombin-activated platelets) showed that hirudin (SO3-) and BJC efficiently inhibit the thrombin formation (43% and 84%, respectively). Taken together, our results suggest that proexosite I blockage decreases the productive recognition of prothrombin as substrate by factor Xa-factor Va complex and prothrombinase complex. Furthermore, data obtained with human platelets suggest that proexosite I may play an important role in the physiological activation of prothrombin.

Carbohydrate moieties on the procofactor factor V, but not the derived cofactor factor Va, regulate its inactivation by activated protein C.

Factor V (FV) is a single-chain plasma protein containing 13-25% carbohydrate by mass. Studies were done to determine if these carbohydrate moieties altered the activated protein C (APC)-catalyzed cleavage and inactivation of both FV and the cofactor which results from its activation by alpha-thrombin, factor Va(IIa) (FVa(IIa)). Treatment of purified FV with N-glycanase and neuraminidase under nonprotein-denaturing conditions removed approximately 20-30% of the carbohydrate from the heavy chain region of the molecule. When glycosidase-treated FV was analyzed in an aPTT (activated partial thromboplastin time)-based APC sensitivity assay, the APC sensitivity ratio (APC-SR) increased from 2.34 to 3.33. In contrast, when glycosidase-treated FV was activated with alpha-thrombin, the addition of the resulting FVa(IIa) to the plasma-based APC sensitivity assay produced no substantial increase in the APC-SR. Additional functional analyses of the APC-catalyzed inactivation of FVa(IIa) in an assay consisting of purified components indicated that both glycosidase-treated and untreated FVa(IIa) expressed identical cofactor activities and were inactivated at identical rates. Analyses of the APC-catalyzed cleavage of glycosidase-treated FV at Arg(306), the initial cleavage site, revealed a 10-fold rate increase when compared to untreated FV. In contrast, and consistent with functional assays, similar analyses of FVa(IIa), derived from those FV species, revealed near-identical rates of APC-catalyzed cleavage at both the Arg(506) and Arg(306)sites. These combined results indicate that N-linked carbohydrate moieties play a substantial role in the APC-catalyzed cleavage and inactivation of FV but not FVa(IIa) at position Arg(306) and that the Arg(306) cleavage sites of FV and FVa(IIa) are distinct substrates for APC.

Mechanism of factor Va inactivation by plasmin. Loss of A2 and A3 domains from a Ca2+-dependent complex of fragments bound to phospholipid.

The coagulation cofactor Va (FVa) is a noncovalent heterodimer consisting of a heavy chain (FVaH) and a light chain (FVaL). Previously, the fibrinolytic effector plasmin (Pn) has been shown to inhibit FVa function. To understand this mechanism, the fragmentation profile of human FVa by Pn and the noncovalent association of the derived fragments were determined in the presence of Ca(2+) using anionic phospholipid (aPL)-coated microtiter wells and large (1 microm) aPL micelles as affinity matrices. Following Pn inactivation of aPL-bound FVa, a total of 16 fragments were observed and their NH(2) termini sequenced. These had apparent molecular weights and starting residues as follows (single letter abbreviation is used): 50(L1766), 48(L1766), 43(Q1828), 40(Q1828), 30(S1546), 12(T1657), and 7(S1546) kDa from FVaL; and 65(A1), 50(A1), 45(A1), 34(S349), 30(L94), 30(M110), and 3 small <5(W457, W457, and K365) kDa from FVaH. Of these, 50(L1766), 48(1766), 43(Q1828), and 40(Q1828) spanning the C1/C2 domains, and 30(L94), but not the similar 30(M110), positioned within the A1 domain remained associated with aPL. These were detected antigenically during Pn- or tissue plasminogen activator-mediated lysis of fibrin clot formed in plasma. Chelation by EDTA dissociated the 30(L94)-kDa fragment, which was observed to associate with intact FVaL upon recalcification, indicating that the Leu-94 to Lys-109 region of the A1 domain plays a critical role in the FVaL and FVaH Ca(2+)-dependent association. By using domain-specific monoclonal antibodies and an assay for thrombin generation, loss of FVa prothrombinase function was coincident with proteolysis at sites in the A2 and A3 domains resulting in their dissociation. Inactivation of FV or FVa by Pn was independent of the thrombophilic R506Q mutation. These results identify the molecular composition of Pn-cleaved FVa that remains bound to membrane as largely A1-C1/C2 in the presence of Ca(2+) and suggest that Pn inhibits FVa by a process involving A2 and A3 domain dissociation.

Homocysteine inhibits inactivation of factor Va by activated protein C.

We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by alpha-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min(-1) at 0, 0.03, 0.1, 1 mm homocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm beta-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [(35)S]homocysteine (10-450 micrometer) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with beta-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).

C4b-binding protein protects coagulation factor Va from inactivation by activated protein C.

We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.

The second laminin G-type domain of protein S is indispensable for expression of full cofactor activity in activated protein C-catalysed inactivation of factor Va and factor VIIIa.

Vitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasma-based assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.

A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition.

The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-peptide), leaving behind the membrane-bound A1.LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (Kd = 7 nM), suggesting that the membrane-bound LC.A1 product would act to sequester APC. This inhibitory interaction (LC.A1.APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Plasma lipoproteins support prothrombinase and other procoagulant enzymatic complexes.

The prothrombinase complex (factor [F]Xa, FVa, calcium ions, and lipid membrane) converts prothrombin to thrombin (FIIa). To determine whether plasma lipoproteins could provide a physiologically relevant surface, we determined the rates of FIIa production by using purified human coagulation factors, and isolated fasting plasma lipoproteins from healthy donors. In the presence of 5 nmol/L FVa, 5 nmol/L FXa, and 1.4 micromol/L prothrombin, physiological levels of very low density lipoprotein (VLDL) (0.45 to 0.9 mmol/L triglyceride, or 100 to 200 micromol/L phospholipid) yielded rates of 2 to 8 nmol Flla x L(-1) x s(-1) in a donor-dependent manner. Low density lipoprotein (LDL) and high density lipoprotein (HDL) also supported prothrombinase but at much lower rates (< or =1.0 nmol FIIa x L(-1) x s[-1]). For comparison, VLDL at 2 mmol/L triglyceride yielded approximately 50% the activity of 2X10(8) thrombin-activated platelets per milliliter. Although the FIIa production rate was slower on VLDL than on synthetic phosphatidylcholine/phosphatidylsenne vesicles (approximately 50 nmol FIIa x L(-1) x s[-1]), the prothrombin Km values were similar, 0.8 and 0.5 micromol/L, respectively. Extracted VLDL lipids supported rates approaching those of phosphatidylcholine/phosphatidylserine vesicles, indicating the importance of the intact VLDL conformation. However, the presence of VLDL-associated, factor-specific inhibitors was ruled out by titration experiments, suggesting a key role for lipid organization. VLDL also supported FIIa generation in an assay system comprising 0.1 nmol/L FVIIa; 0.55 nmol/L tissue factor; physiological levels of FV, FVIII, FIX, and FX; and prothrombin (3 nmol/L FIIa x L(-1) x s[-1]). These results indicate that isolated human VLDL can support all the components of the extrinsic coagulation pathway, yielding physiologically relevant rates of thrombin generation in a donor-dependent manner. This support is dependent on the intact lipoprotein structure and does not appear to be regulated by specific VLDL-associated inhibitors. Further studies are needed to determine the extent of this activity in vivo.