ABSTRACT
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.
Subject(s)
Factor Va/chemistry , Factor Xa/chemistry , Peptides/chemistry , Proteolysis , Factor Va/antagonists & inhibitors , Factor Va/genetics , Factor Va/metabolism , Factor Xa/genetics , Factor Xa/metabolism , Humans , Peptides/genetics , Peptides/metabolism , Protein Binding , Protein Structure, TertiaryABSTRACT
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.
Subject(s)
Anticoagulants/pharmacology , Aptamers, Nucleotide/pharmacology , Blood Coagulation/drug effects , Factor V/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Amino Acid Sequence , Anticoagulants/chemistry , Anticoagulants/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Pairing , Binding Sites , COVID-19/blood , Cell Membrane/chemistry , Cell Membrane/metabolism , Factor V/chemistry , Factor V/genetics , Factor V/metabolism , Factor Va/chemistry , Factor Va/genetics , Factor Va/metabolism , Heparin, Low-Molecular-Weight/chemistry , Heparin, Low-Molecular-Weight/metabolism , Humans , Immune Sera/chemistry , Immune Sera/metabolism , Models, Molecular , Nucleic Acid Conformation , Protamines , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , SELEX Aptamer Technique , Substrate Specificity , COVID-19 Drug TreatmentABSTRACT
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.
Subject(s)
Factor V/metabolism , Factor Va/metabolism , Factor Xa/metabolism , Phospholipids/metabolism , Prothrombin/metabolism , 1-Carboxyglutamic Acid/chemistry , 1-Carboxyglutamic Acid/genetics , 1-Carboxyglutamic Acid/metabolism , Binding, Competitive/drug effects , Cell Line , Epidermal Growth Factor/chemistry , Epidermal Growth Factor/genetics , Epidermal Growth Factor/metabolism , Factor V/chemistry , Factor Va/antagonists & inhibitors , Factor Va/chemistry , Factor Xa/chemistry , Humans , Phosphatidylcholines/pharmacology , Phosphatidylserines/pharmacology , Phospholipids/chemistry , Protein Binding/drug effects , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/genetics , Prothrombin/antagonists & inhibitors , Prothrombin/chemistry , Prothrombin/physiology , Sequence Deletion/drug effects , Static Electricity , Substrate Specificity/drug effects , Thromboplastin/chemistry , Thromboplastin/metabolismABSTRACT
Coagulation Factor V is an essential component of the prothrombinase complex, which activates the zymogen prothrombin to thrombin. A patient was described who developed a Factor V inhibitor that neutralized the procoagulant activity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we have found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant activity of Factor Va, implying that the neutralization resulted from specific binding to the C2 domain. We have previously demonstrated that deletion of the C2 domain results in loss of procoagulant activity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserine-specific binding of Factor V. Conversely, preincubation of Factor Va with procoagulant phospholipids protected the cofactor from inactivation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant activity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex.
Subject(s)
Factor V/antagonists & inhibitors , Hemorrhagic Disorders/immunology , Phosphatidylserines/metabolism , Antigen-Antibody Reactions , Base Sequence , Blood Coagulation , DNA Mutational Analysis , Epitopes , Factor V/chemistry , Factor V/immunology , Factor Va/antagonists & inhibitors , Factor Va/immunology , Factor Va/metabolism , Humans , In Vitro Techniques , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Precipitin Tests , Recombinant Proteins/chemistry , Sequence DeletionABSTRACT
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.
Subject(s)
Anticoagulants/therapeutic use , Cardiovascular Diseases/drug therapy , Coronary Thrombosis/drug therapy , Fibrinolytic Agents/therapeutic use , Platelet Aggregation Inhibitors/therapeutic use , Anticoagulants/pharmacology , Blood Coagulation/drug effects , Blood Platelets/drug effects , Cardiovascular Diseases/etiology , Cardiovascular Diseases/physiopathology , Cardiovascular Diseases/prevention & control , Coronary Thrombosis/etiology , Coronary Thrombosis/physiopathology , Coronary Thrombosis/prevention & control , Factor VIIa/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Factor Xa/chemistry , Factor Xa Inhibitors , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacology , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/adverse effects , Platelet Aggregation Inhibitors/chemistry , Structure-Activity Relationship , Thrombin/antagonists & inhibitors , Venous Thrombosis/drug therapy , Venous Thrombosis/etiology , Venous Thrombosis/physiopathologyABSTRACT
Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide-(311-325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa-1-stage coagulation assays in normal and in protein S-depleted plasma with 50% inhibition at 13 microM peptide. In a system using purified clotting factors, peptide-(311-325) inhibited APC-catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 microM peptide. However, peptide-(311-325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]alpha 1-antitrypsin. Peptide-(311-325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 microM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose-dependent manner to immobilized peptide-(311-325). Peptide-(311-315) inhibited the binding of factor Va to immobilized APC or factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Factor Va/antagonists & inhibitors , Peptide Fragments/pharmacology , Protein C/pharmacology , Amino Acid Sequence , Anticoagulants , Binding Sites , Factor Va/metabolism , Factor Xa Inhibitors , Fibrinogen/metabolism , Humans , Molecular Sequence Data , Partial Thromboplastin Time , Peptide Fragments/metabolism , Protein C/antagonists & inhibitors , Protein C/metabolism , Thrombin/pharmacology , Thromboplastin/antagonists & inhibitorsABSTRACT
When whole blood is allowed to clot in vitro, factor V is rapidly activated to factor Va which is subsequently inactivated. We developed two monoclonal anti-protein C antibodies, one of which inhibits protein C activation and the other inhibits protein C activity. The addition of either antibody to blood before clotting in vitro significantly inhibited the inactivation of factor Va, confirming the essential role of protein C in mediating the rapid inactivation of factor Va.
Subject(s)
Factor Va/antagonists & inhibitors , Protein C/physiology , Thrombosis/physiopathology , Antibodies, Monoclonal , Humans , In Vitro Techniques , Protein C/immunology , Time FactorsABSTRACT
A simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 +/- 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p less than 0.001). Also, a similar correlation (r = 0.93, p less than 0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.
Subject(s)
Blood Chemical Analysis/methods , Protein C/analysis , Anticoagulants , Crotalid Venoms , Evaluation Studies as Topic , Factor Va/antagonists & inhibitors , HumansABSTRACT
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.
Subject(s)
Crotalid Venoms/pharmacology , Enzyme Inhibitors/pharmacology , Factor V/metabolism , Factor Va/antagonists & inhibitors , Factor Xa/metabolism , Prothrombin/metabolism , Thrombin/biosynthesis , Binding Sites/drug effects , Blood Platelets/physiology , Enzyme Activation/drug effects , Factor V/chemistry , Factor Xa/chemistry , Hirudins/pharmacology , Humans , Kinetics , Liposomes , Macromolecular Substances , Peptide Fragments/pharmacology , Phosphatidylcholines/pharmacology , Phosphatidylserines/pharmacology , Phospholipids/pharmacologyABSTRACT
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.
Subject(s)
Factor VIIIa/agonists , Factor Va/antagonists & inhibitors , Intercellular Signaling Peptides and Proteins , Protein C/pharmacology , Protein S/chemistry , Anticoagulants/chemistry , Anticoagulants/metabolism , Anticoagulants/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Laminin/chemistry , Partial Thromboplastin Time , Protein C/metabolism , Protein S/metabolism , Protein S/pharmacology , Protein Structure, Tertiary , Proteins/chemistry , Proteins/pharmacology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacology , Sex Hormone-Binding Globulin/chemistry , Sex Hormone-Binding Globulin/pharmacologyABSTRACT
The effects of annexin V on the anticoagulant activity of activated protein C (APC) and protein S were examined. Although annexin V did not influence the amidolytic potential of APC, it inhibited both APC and protein S function in a factor Va inactivation assay. Competition experiments demonstrated that annexin V inhibits protein S binding to phospholipid vesicles in a dose-dependent manner. These results demonstrate that annexin V effectively interferes with the anticoagulant arm of the hemostatic system.
Subject(s)
Annexin A5/pharmacology , Blood Coagulation/drug effects , Factor Va/antagonists & inhibitors , Lipids/blood , Protein C/antagonists & inhibitors , Protein S/antagonists & inhibitors , Annexin A5/metabolism , Humans , Membranes, Artificial , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Phospholipids/metabolism , Protein Binding , Protein S/metabolism , Structure-Activity RelationshipABSTRACT
The expression of tissue factor pathway inhibitor (TFPI) was investigated in sinusoidal endothelial cells in the liver and endothelial cells in the lung. Northern blot analysis revealed that TFPI mRNA was expressed in the lung, but minimal in the liver. Also, immunohistochemical examination showed that TFPI was not expressed on the sinusoidal endothelial cells in contrast to marked expression on endothelial cells in the lung, suggesting that anticoagulant activity to inhibit blood coagulation induced by tissue factor is reduced in the hepatic sinusoids compared to the microvessels of other organs. When recombinant human TFPI was intravenously injected in rats, it disappeared rapidly from the circulation, but was detected by electron microscopy on the surface of sinusoidal endothelial cells and microvilli of hepatocytes in the space of Disse. In these rats, the TFPI reappeared in the circulation following an intravenous injection of heparin sodium with reduced immunohistochemical staining of the TFPI on hepatic sinusoidal walls. It is concluded that exogenous TFPI can increase anticoagulant activity on the hepatic sinusoidal walls by binding to heparinoids on the cell surface. It may act effectively even in the hepatic sinusoids with damaged endothelial cells.
Subject(s)
Anticoagulants/metabolism , Factor Va/antagonists & inhibitors , Fibrinolytic Agents/metabolism , Lipoproteins/metabolism , Liver/metabolism , Serine Proteinase Inhibitors/metabolism , Animals , Anticoagulants/pharmacology , Blotting, Northern , Fibrinolytic Agents/pharmacology , Humans , Lipoproteins/genetics , Lipoproteins/pharmacology , Liver/ultrastructure , Liver Transplantation/pathology , Lung/metabolism , Necrosis , RNA, Messenger/metabolism , Rats , Rats, Inbred F344 , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/pharmacologyABSTRACT
Factor V Leiden is characterised by a point mutation which prevents the physiologic inhibition of activated factor V by activated protein C (Activated Protein C Resistance). This mutation is now considered as the most frequent inherited thrombophilic disorder. It is found in about 20% of patients with venous thrombotic disease, far before ATIII, Protein C and S deficiency. Its prevalence (4% in the general belgian population, 1 to 15% in Europa) allows frequent associations with other thrombophilic disorders, inherited or acquired, such as contraceptive pill. Biological testing are now ready for screening, but the opportunity of a systematic evaluation in front of a risk situation remains a matter to discussion.
Subject(s)
Factor V/genetics , Factor Va/antagonists & inhibitors , Point Mutation/genetics , Protein C/physiology , Thrombosis/genetics , Europe/epidemiology , Humans , Mass Screening , Prevalence , Risk Factors , Thrombosis/epidemiology , Thrombosis/prevention & controlABSTRACT
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.
Subject(s)
Anticoagulants/therapeutic use , Endothelium, Vascular/drug effects , Fluorobenzenes/therapeutic use , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Sulfonamides/therapeutic use , Venous Thromboembolism/prevention & control , Animals , Blood Coagulation/drug effects , Clinical Trials as Topic , Endothelium, Vascular/metabolism , Factor Va/antagonists & inhibitors , Fluorobenzenes/pharmacology , Gene Expression Regulation/drug effects , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Models, Animal , Pyrimidines/pharmacology , Rosuvastatin Calcium , Sulfonamides/pharmacology , Thrombin/metabolism , Thrombomodulin/genetics , Thrombomodulin/metabolism , Thromboplastin/genetics , Thromboplastin/metabolism , Venous Thromboembolism/bloodABSTRACT
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.
Subject(s)
Anticoagulants/administration & dosage , Drugs, Investigational/administration & dosage , Thrombosis/drug therapy , Administration, Oral , Antithrombin III/administration & dosage , Clinical Trials as Topic , Factor VIIIa/antagonists & inhibitors , Factor VIIa/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Humans , Lipoproteins/administration & dosage , Thrombin/antagonists & inhibitors , Thrombosis/bloodABSTRACT
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.
Subject(s)
Factor Va/antagonists & inhibitors , Heparin/pharmacology , Protein C/physiology , Blotting, Western , Catalysis , Crystallography, X-Ray , Kinetics , Models, MolecularABSTRACT
An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of protein S toward factor Va inactivation by activated protein C (APC). The initial rate of endothelial cell-mediated factor Va inactivation was 21.7 pM factor Va/50 pM APC min-1, which could be enhanced twice at a protein S concentration of 5 nM. This increase appeared to be specific for protein S because it could be inhibited by C4b-binding protein and polyclonal antibodies against protein S. Furthermore, thrombin-cleaved protein S did not accelerate factor Va inactivation by APC on endothelial cells. The binding of 125I-protein S to endothelial cells was time-dependent, specific, saturable, and required the presence of calcium ions. Scatchard analysis revealed (8.0 +/- 0.3) x 10(5) binding sites per cell with an apparent Kd of 24.4 +/- 2.2 nM. To study the physiological importance of the binding of protein S to human endothelial cells, seven monoclonal antibodies were examined for their ability to influence the protein S cofactor activity and binding capacity. Monoclonal antibodies directed against the gamma-carboxyglutamic acid domain and the thrombin-sensitive region of protein S completely inhibited the protein S cofactor function in factor Va inactivation by APC on HUVECs. These monoclonal antibodies also inhibited 125I-protein S binding to HUVECs. Another monoclonal antibody, directed against an epitope on the third and/or fourth epidermal growth factor-like region, did not influence either protein S cofactor activity or binding of protein S to HUVECs. We conclude that binding of protein S to HUVECs is essential for the expression of its cofactor activity for APC. At least two regions in protein S, the gamma-carboxyglutamic acid domain and the thrombin-sensitive region, are involved in the expression of cofactor activity.
Subject(s)
Endothelium, Vascular/metabolism , Protein C/metabolism , Protein S/metabolism , Antibodies, Monoclonal , Cells, Cultured , Endothelium, Vascular/cytology , Factor Va/antagonists & inhibitors , Humans , Iodine Radioisotopes , Protein Binding , Substrate SpecificityABSTRACT
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.
Subject(s)
Activated Protein C Resistance/blood , Autoantibodies/blood , Factor V/immunology , Venous Thrombosis/blood , Adult , Blood Coagulation Tests , Chromatography, Affinity , Drug Resistance , Epitopes/isolation & purification , Factor V/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Female , Humans , KineticsABSTRACT
Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify the regions on the surface that mediate anticoagulant activity, 26 synthetic peptides were prepared representing 90% of the human protein C heavy chain primary structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404) specifically inhibited APC activity in activated partial thromboplastin time and Xa-1-stage coagulation assays in normal, in protein S-depleted and Factor VIII-deficient plasma with 50% inhibition at 5 microM peptide. Polyclonal antibodies raised against this peptide and immunoaffinity-purified on a protein C-Sepharose column inhibited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, protein S-depleted, and Factor VIII-deficient plasma with half-maximal inhibition at 30 nM anti-(390-404) antibody. Neither the peptide-(390-404) nor the anti-(390-404) antibodies inhibited APC amidolytic activity or the reaction of APC with recombinant [Arg358] alpha 1-antitrypsin. Furthermore, in a purified system, peptide-(390-404) inhibited APC-catalyzed inactivation of Factor Va in the presence as well as in the absence of phospholipids with 50% inhibition at 4 microM peptide. These data suggest that the region containing residues 390-404 in APC is essential for anticoagulant activity and is available to interact with antibodies or with other proteins such as the macromolecular substrates Factors Va or VIIIa.
Subject(s)
Blood Coagulation , Protein C/physiology , Amino Acid Sequence , Catalysis , Factor VIIIa/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Humans , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/genetics , Peptide Fragments/physiology , Protein C/genetics , Sequence AlignmentABSTRACT
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.