Interaction of factor VIII with phospholipids: role of composition and negative charge.

Radiolabelled human anti-FVIII:C antibody was affinity-purified according to its ability to bind to factor VIII-phospholipid (FVIII-PL) complexes, yielding a fraction directed against the phospholipid binding-site (PL-site antibody). This antibody was used as a specific probe for FVIII binding to PL vesicles containing a variety of natural and synthetic PLs. Of purified PLs tested for FVIII binding, phosphatidyl serine (PS) and phosphatidic acid (PA) were highly active, phosphatidyl inositol (PI) much less so, and both phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) inactive: the apparent dissociation constant (Kd app) for FVIII binding to PS:PC vesicles showed a strong dependence on PS content. Free-flow electrophoresis of vesicles confirmed FVIII binding to PS:PC required both net negative charge and specific head-group: neither PS vesicles given a positive charge with stearylamine nor PC vesicles made negative with dicetyl phosphate bound FVIII. It is concluded that the negative charge required for FVIII binding must be presented on the phospholipid surface in the correct orientation: phosphatidyl serine supplies this charge in coagulant-active PL preparations.     

L-asparaginase treatment reduces the anticoagulant potential of the protein C system without affecting vitamin K-dependent carboxylation

The changes in plasma levels of the vitamin K-dependent natural anticoagulants protein C (PC) and protein S (PS) and procoagulant factors II, IX and X were evaluated in 8 adult patients during treatment with L-asparaginase (L-ase i.v. 120,000 U/m² over 10 days). PC anticoagulant activity and factor IX, X and II coagulant activity decreased proportionally to their half-lives to a nadir of 50–60% of pretreatment values after 2–5 L-ase infusions, suggesting that inhibition of protein synthesis rather than consumption is the main mechanism responsible for the observed changes. Free PS antigen levels declined at a rate similar to total PS antigen, reaching a nadir of 56% of pretreatment values after 3 L-ase infusions; however, due to C4b-binding protein levels higher than total PS levels (p <0.05), they were constantly lower than the corresponding total PS antigen levels (0.05 < p <0.001). This implicates that total PS antigen levels cannot be taken as an indicator of PS activity. No differences between the antigenic levels and the anticoagulant activities of PC and free PS could be observed suggesting that L-ase does not affect the mechanisms of vitamin K-dependent carboxylation of Gla-residues. The faster rate of decline of PC and PS activities relative to that of factor II may be responsible for the onset of an hypercoagulable state during the early phase of L-ase treatment.     

The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis

The contribution of the factor Va C1 domain (fVa-C1) to assembly of the prothrombinase complex has not been previously investigated. The homologous fVa-C2 domain contains a binding site for phosphatidylserine (PS) that includes the indole moieties of Trp(2063)/Trp(2064) at the apex of spike-1. In order to investigate the structure and function of fVa-C1 a molecular model was constructed based on the structure of fVa-C2. The aromatic and hydrophobic side chains of Tyr (1956) /Leu (1957) in fVa-C1 are located at the predicted apex of spike-3. Exposed charged and hydrophobic residues in fVa-C1 were changed to alanine in clusters of 1-3 mutations per construct.The resultant 20 mutants were expressed in COS cells and screened for binding to immobilized PS and prothrombinase activity on phospholipid vesicles containing either 25% or 5% PS. Two mutants, (Y1956,L1957)A, and (R2023,R2027)A showed both decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS. The interaction of purified (Y1956,L1957)A with phospholipid vesicles was studied using fluorescence resonance energy transfer and prothrombinase assays. The affinity of (Y1956,L1957)A binding to 25% PS membranes was reduced 12-fold compared to rHFVa. Prothrombin activation in the presence of (Y1956,L1957)A was markedly impaired on phos-pholipid vesicles containing 10% or less PS. We conclude that solvent exposed hydrophobic and aromatic amino acids in both fVa-C1 and fVa-C2 contribute to the interaction of factor V with PS membranes.     

Phospholipid vesicles interfere with the binding of antibody fragments to the light chain of factor VIII

Factor VIII binds to phospholipid membranes through the C2 domain (S2173-Y2332). Residues M2199, F2200, L2251, L2252, V2223, W2313 and V2314 at the tips of beta-hairpins and loops are thought to contribute to phospholipid membrane binding. Similarly, residues in the C2 domain of the homologous protein factor V forma phospholipid binding site, but residues in the A3 and C1 domains are also thought to contribute to membrane binding. Phage display technology was previously used to isolate factor VIII light chain specific single-chain variable domain fragments (scFv) from patients with factor VIII inhibitors. Phospholipid vesicles inhibited the binding of factor VIII to scFvs WR1 and WR16 (epitope : E2181-M2199) with half saturation values of 23 and 47 muM respectively. The single point mutant F2200A factor VIII light chain bound to WR1 and WR16 with a much lower affinity than wild type protein suggesting that residue F2200 is also included in the epitopes of these scFvs. Binding of factor VIII to C2-specific scFvs WR13 and EL14 (epitope : K2207-M2321) was not inhibited by phospholipid vesicles. Consistent with this, F2200A factor VIII light chain bound to these scFvs with the same affinity as the wild type protein. However, phospholipid vesicles also inhibited the binding of factor VIII to the A3-C1-specific scFvs KM36 (epitope : Q1778-D1840) and KM38 (epitope : S1690-N1777 and/or V1841-N2172) with half saturation values of 84 and 165 microM, respectively, suggesting that the A3 and/or C1 domains may contribute to membrane binding of the cofactor.     

Binding of annexin V to a human ovarian carcinoma cell line (OC-2008). Contrasting effects on cell surface factor VIIa/tissue factor activity and prothrombinase activity.

Proteins of the annexin/lipocortin family bind tightly to anionic phospholipids and platelets and act as in vitro anticoagulants. Annexins may be useful as tools to study the availability of anionic phospholipids on cell surfaces and their role in the regulation of blood coagulation. In the present study, we investigated the binding of annexin V (placental anticoagulant protein I) to a human ovarian carcinoma cell line, OC-2008, that constitutively expresses surface membrane tissue factor activity. Binding of annexin V to cell monolayers was calcium-dependent, specific, saturable and reversible; Scatchard analysis indicated a single class of binding sites with an apparent Kd of 9.4 +/- 3.1 nM and 5.2 +/- 1 x 10(6) sites per cell. Binding was completely inhibited by phospholipid vesicles containing phosphatidylserine, but was not inhibited by vesicles containing phosphatidylcholine. Annexin V inhibited the cell surface-dependent activity of prothrombinase complex, but did not inhibit the activity of the factor VIIa/tissue factor complex. In conclusion, these results suggest that anionic phospholipid is present on the extracellular face of OC-2008 cells; this anionic phospholipid is functionally important for the activity of the prothrombinase complex, but the importance of anionic phospholipid for the cell surface factor VIIa/tissue factor functional activity is unclear.     

Comparison of the abilities of synthetic and platelet-derived membranes to enhance thrombin formation

The relative abilities of platelet-derived membranes and synthetic phospholipid vesicles to enhance the prothrombinase-catalyzed conversion of prothrombin to thrombin have been determined. For each type of membrane, the maximum amount of thrombin formed as a function of amount of available lipid was measured using a chromogenic substrate assay. The lipid concentration at which the amount of thrombin formed began to exceed that formed in the absence of lipid (critical phospholipid concentration) was used to compare the surfaces′ abilities to support thrombin formation. For platelet derived membranes and for equimolar, charged-lipid/phosphatidylcholine (PC) vesicles, the critical concentrations increased in the following order: platelet-derived membranes phosphatidylserine (PS) phosphatidic acid (PA) « monomethyl PA and monoethyl PA « phosphatidylinositol and phosphatidylglycerol. For mixed anionic/ neutral lipid vesicles above their phase transitions, measured critical concentrations were relatively insensitive to changes in lipid acyl chains, the neutral lipid component, and membrane curvature but were sensitive to changes in the anionic lipid content of the mixtures. Comparison of these data suggested that equimolar PS/PC and PA/PC vesicles can emulate reasonably well the thrombin-generating ability of platelet-derived membranes.     

Binding of [3H]-dihydroalprenolol and [3H]-acetobutolol to human blood platelets is not related to occupancy of beta-adrenoceptors

Binding of {³H}-dihydroalprenolol to human platelet lysates is inhibited by (±)-propranolol and (±)-butoxamine, but less effectively by (±) practolol. (−)-Isoprenaline causes no significant inhibition of binding where stimulation of adenylate cyclase can be shown. Binding of {³H}-acetobutolol is also inhibited by (±)-propranolol. “Specific” binding of {³H}-dihydroalprenolol and {³H}-acetobutolol defined by (±) propranolol shows a non-classical saturation curve. 50% maximal binding is observed in the range 15 – 25 mM. The extent of “specific” binding is 2-fold greater for {³H}-dihydroalprenolol. Similar and rapid rates of binding of {³H}-dihydroalprenolol are observed at 4°C and 20°C. No stereoselectivity is observed for inhibition of {³H}-dihydroalprenolol binding by (+) and (−)-propranolol. Binding of {³H}-dihydroalprenolol and {³H}-acetobutolol may relate to the lipophilic character of these radioligands and does not represent interaction with β-adrenoceptors.     

The mechanism of the fibrinogen-thrombin reaction

The kinetics of the thrombin-catalysed release of fibrinopeptide A from fibrinogen, modified fibrinogen and various fragments of fibrinogen has been investigated in an attempt to elucidate the structural elements of fibrinogen which define its interaction with thrombin. These elements have been found to be contained within the structure formed by the first 51 amino acid residues of the A-chain. It is proposed that the binding which is of fundamental importance takes place within the sequence 1–23 of this chain and may even be localised to the sequence 8–16. This binding is strengthened, either directly or indirectly, by structures within the sequence 34–44 and to a lesser extent by structures within the sequence 45–51.     

Interaction of fibrinogen with mercury

The interaction of bovine fibrinogen with mercuric chloride was studied. Gel filtration on Sephadex G-25 revealed that fibrinogen bound twice the amount of mercury such as fibrin or fibrin monomers (8.8, 4.5, and 3.4 μg Hg²⁺ ions/ mg protein, respectively). Fibrinogen complexed with mercury or in the presence of Hg²⁺ ions at concentration above 10⁻⁶ M was clotted by thrombin more effectively than in the control system which was devoid of this metal. Reaggregation of the purified fibrin monomers was not affected by mercury.     

GM1 ganglioside partially rescues cultured dopaminergic neurons from MPP-induced damage: dependence on initial damage and time of treatment

GM1 ganglioside is believed to be important in promoting the recovery of neurons from injury. The present study assesses the ability of GM1 to repair or prevent the damage of dopamine neurons caused by the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺). Treatment of mesencephalic cell cultures with 2.5 μM MPP⁺ resulted in the loss of 30% of tyrosine hydoxylase (TH) immunoreactive neurons. In contrast, cultures administered 100 μM GM1 ganglioside for 3 days after toxin treatment contained nearly control numbers of TH⁺ neurons (97%). This reparative effect of GM1 was reflected in parallel increases in TH enzyme activity, dopamine and dopac levels. Cultures sustaining greater insult from higher doses of MPP⁺ (5.0–10.0 μM) did not benefit from ganglioside treatment, suggesting that rescue by GM1 depended on the degree of initial damage to cells. Moreover, the timing of ganglioside treatment was critical; pretreatment with GM1 alone did not prevent or attenuate the damage caused by subsequent incubation in 2.5 μM MPP⁺.     

Interaction of fibrinogen with fluorescein isothiocyanate

The effect of increasing labelling with fluorescein isothiocyanate /FITC/ on the properties of pig fibrinogen was investigated. Conjugation of fibrinogen with 2–3 fluorescein residues per molecule resulted in essentially unchanged clottability as compared to control fibrinogen. No changes in molecular size due to covalent aggregation were observed by SDS gel electrophoresis. Fibrinogen conjugated with about 20 FITC residues per molecule did not form any visible clot. About 50% of the label was bound to the Aα chain, 33% to the Bβ, and only 17% to the α chain.     

Fibrinogen bondy : A new case of dysfibrinogenemia. Isolation of the abnormal fibrinogen molecules

In a 81 year old healthy woman, gross abnormalities of fibrin formation led to the discovery of an abnormal fibrinogen named fibrinogen Bondy. Clottability of purified fibrinogen Bondy was only 53% compared to 95–98% for normal fibrinogen. Functional studies revealed (i) delayed coagulation by thrombin and batroxobin (Reptilase), (ii) incomplete release of fibrino-peptides A and B, (iii) poor fibrin monomer aggregation, (iv) delayed fibrin proteolysis by plasmin. Electrophoretic mobility of fibrinogen Bondy, its three chains and the products of fibrin cross-linking, was normal. Fibrinogen NH₂-terminal residues of fibrinogen Bondy were found to be normal. The presence of Ala, in addition to Gly and Tyr in the fibrin clot and its supernatant, showed that a part of fibrinogen molecules was not clotted, i.e. either copolymerised with fibrin or remaining in solution. Gel filtration of the supernatant allowed the separation of both soluble complexes and fibrinogen. This fibrinogen population was shown to be unclottable by thrombin and to inhibit clotting of normal fibrinogen.     

Decreased erythrocyte and platelet phospholipids and fatty acids in juvenile neuronal ceroid-lipofuscinosis (Batten disease)

Total cellular and phospholipid fatty acids were analyzed in erythrocytes and platelets from six patients with juvenile neuronal ceroid-lipofuscinosis (Spielmeyer-Vogt, Batten disease, JNCL). The results were compared to those of age-matched controls. The amounts of total fatty acid and the phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) were significantly decreased in patients when related to cellular protein. The reductions in total fatty acids were 27% in erythrocytes and 50% in platelets. Erythrocyte PC reduction was 36%, PE was 44% and PS 27%. There were no major qualitative differences in the phospholipid fatty acids, suggesting that the fatty acid composition of the reduced phospholipid was normal, and that there is a generalized phospholipid deficiency in these cells in JNCL. This was not related to nutritional status. The pathogenesis of Batten disease may be related to abnormal membrane function resulting from this marked phospholipid deficiency.     

Fibrinogen Detroit - an abnormal fibrinogen with non-functional NH2-terminal polymerization domain

Fibrinogen Detroit was found to have affinity for thrombin activated fibrinogen-Sepharose conjugates prepared from normal fibrinogen. On the other hand, thrombin activated fibrinogen-Sepharose conjugates prepared from fibrinogen Detroit did not, to any appreciable extent, bind normal fibrinogen nor the plasmic degradation product, Fragment D. It is concluded that the N-DSK domain in fibrinogen Detroit, containing the mutation (Aα 19 Arg → Ser), is abnormal with regard to binding whereas the Fragment D domain has normal binding properties.     

The dimeric Aalpha chain composition of dysfibrinogenemic molecules with mutations at Aalpha 16

In the last stage of fibrinogen synthesis, two Aalpha-Bbeta-gamma half-molecules are disulfide linked in their N-terminal regions to form a dimeric fibrinogen molecule. It is not known whether intracellular hepatocyte assembly of fibrinogen half-molecules occurs randomly or is a directed process. One analysis based on partitioning of coagulable components of fibrinogen from a heterozygous dysfibrinogenemic subject having a mutation at the thrombin cleavage site (Fibrinogen Louisville, Aalpha16 R-->H), suggested that only homodimeric molecules containing two normal fibrinopeptides A (FPA, FPA) or two abnormal fibrinopeptides A (FPA*, FPA*) were present in plasma, implying that fibrinogen dimer assembly is directed. The same type of analyses on Fibrinogen Birmingham (Aalpha16 R-->H) indicated that there were heterodimers as well as homodimers, suggesting that fibrinogen dimer assembly is random. To examine this question more directly, the composition of fibrinogen molecules from seven dysfibrinogenemic families with either R-->C (four) or R-->H (three) Aalpha16 mutations was determined. Following treatment with Atroxin to release normal FPA from fibrinogen, N-terminal disulfide knot ('N-DSK') cleavage fragments were prepared and subsequently separated by SDS-PAGE to resolve 'N-DSK' components with two FPA*'s (N-DSK homodimer), one FPA* (des A N-DSK heterodimer), or no FPA's (des AA N-DSK homodimer). Fibrinogen from subjects whose molecules contained both normal and abnormal Aalpha chains, yielded a heterodimeric des A N-DSK derivative, as well as smaller amounts of homodimeric N-DSK and des AA N-DSK. These results indicate that when both types of Aalpha chain are produced, both Aalpha chain alleles are expressed and the resulting fibrinogen dimers are assembled randomly.     

The dimeric Aα chain composition of dysfibrinogenemic molecules with mutations at Aα 16

In the last stage of fibrinogen synthesis, two Aα-Bβ-γ half-molecules are disulfide linked in their N-terminal regions to form a dimeric fibrinogen molecule. It is not known whether intracellular hepatocyte assembly of fibrinogen half-molecules occurs randomly or is a directed process. One analysis based on partitioning of coagulable components of fibrinogen from a heterozygous dysfibrinogenemic subject having a mutation at the thrombin cleavage site (Fibrinogen Louisville, Aα16 R→H), suggested that only homodimeric molecules containing two normal fibrinopeptides A (FPA, FPA) or two abnormal fibrinopeptides A (FPA, FPA) were present in plasma, implying that fibrinogen dimer assembly is directed. The same type of analyses on Fibrinogen Birmingham (Aα16 R→H) indicated that there were heterodimers as well as homodimers, suggesting that fibrinogen dimer assembly is random. To examine this question more directly, the composition of fibrinogen molecules from seven dysfibrinogenemic families with either R→C (four) or R→H (three) Aα16 mutations was determined. Following treatment with Atroxin to release normal FPA from fibrinogen, N-terminal disulfide knot (‘N-DSK’) cleavage fragments were prepared and subsequently separated by SDS-PAGE to resolve ‘N-DSK’ components with two FPA's (N-DSK homodimer), one FPA (des A N-DSK heterodimer), or no FPA's (des AA N-DSK homodimer). Fibrinogen from subjects whose molecules contained both normal and abnormal Aα chains, yielded a heterodimeric des A N-DSK derivative, as well as smaller amounts of homodimeric N-DSK and des AA N-DSK. These results indicate that when both types of Aα chain are produced, both Aα chain alleles are expressed and the resulting fibrinogen dimers are assembled randomly.     

Apparent involvement of protein kinase C in the central glucoregulatory action of insulin

We have studied the possible involvement of the calcium- and phospholipid/diacylglycerol-dependent enzyme, protein kinase C (PKC) in mediating insulin action in the central nervous system (CNS) by testing the effect of direct activation or blockade of the CNS PKC system on the plasma glucose responses to central insulin injection in mice. Insulin (0.1–1 μg), injected into the CNS, produced rapid transient hypoglycemia. This effect appeared to involve interaction of insulin with specific receptors, since insulin analogs exhibiting diminished receptor binding affinity and peripheral bioactivity compared to the native hormone were much less active (i.e., insulin >>> acetyl 3 insulin > proinsulin > IGF-I) or not active at all (i.e., insulin chain A and B). Central injection of the specific PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.01–0.5 μg), but not the inactive TPA analog, 4-orbol or the unstable synthetic diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), significantly enhanced the hypoglycemic response to co-administered insulin (0.5 μg) or the insulin derivative, acetyl 3 insulin (2.5 μg). Central TPA had no effect on basal glucose levels. Furthermore, central administration of the selective PKC blockers, polymyxin B (PMB, 1–25 μg) or 1-β-galactosylsphingosine (psychosine, 0.5–10 μg) but not their respective inactive analogs, polymyxin E and sphingomyelin, strongly inhibited the hypoglycemic response to insulin (1 μg) or acetyl 3 insulin (5 μg). PMB and psychosine, injected alone had no effect on basal glucose levels. These findings, of a significant enhancement or blockade of the hypoglycemic response to central insulin following direct, selective activation or inhibition, respectively, of the CNS PKC system are consistent with the view that PKC might play a role in the mediation of insulin action in the CNS.     

Ganglioside biosynthesis in Golgi apparatus: new perspectives on its mechanism

The synthesis of gangliosides GM1 and GM2 in intact rat liver Golgi vesicles is stimulated by phosphatidylglycerol as much as or even more than by detergents (Triton X-100 and octyglucoside, respectively). The antibiotic tunicamycin, known as an inhibitor of the N-glycosylation of proteins, strongly inhibits the synthesis of the above gangliosides, in the presence as well as in the absence of the phospholipid. Both phosphatidylglycerol dependence and tunicamycin inhibition disappear when the Golgi vesicles are solubilized by addition of detergents or disrupted by ultrasonication or pretreated with pronase. Transport studies with UDP-[3H]Gal show that tunicamycin blocks the penetration of the sugar nucleotide into the Golgi vesicles in a concentration-dependent manner up to 80%. The results show that tunicamycin inhibits ganglioside biosynthesis by blocking the transport of the nucleotide sugar and not by inhibiting the transferase directly. Studies on glycoprotein-galactosyltransferase with ovalbumin as exogenous acceptor showed that phosphatidylglycerol does not destroy the integrity of the Golgi vesicles. So this phospholipid is an excellent tool for studying ganglioside biosynthesis at optimal transferase activities without solubilizing the Golgi membranes.     

The release of B beta 1-42 from fibrinogen and fibrin by plasmin

The balance between thrombin and plasmin action has been postulated to be an important determinant of thrombosis. Measurement of plasma concentrations of fibrinopeptide A (FPA), which reflect thrombin action on the NH₂-terminal end of the A chain, and of Bβ 1–42 (thrombin-increasable fibrinopeptide B immunoreactivity - TIFPB) which reflect plasmin action on the NH₂-terminal end of the Bβ chain have shown systematic changes in the relative concentrations of the two peptides in thrombotic states. This paper reports kinetic data for TIFPB release by plasmin using fibrinogen, fibrin I monomer, and fibrin I polymer as substrates. For fibrinogen and fibrin I monomer the data fit the Michaelis-Menten equation. Experiments were performed with human proteins in 0.15M Trisbuffered saline at pH 7.4 and at 37°C. With fibrinogen as substrate the K_m was calculated to be 0.87 μM and the V_max 3.75 × 10⁻⁵ M/min/unit of plasmin. With fibrin I monomer as the substrate the K_m was calculated to be 1.25 μM and the V_max 5.5 × 10⁻⁵ M/min/unit of plasmin. With fibrin I polymer as substrate the data did not fit the Michaelis-Menten equation but there appeared to be no dramatic differences in rates from those obtained with the other two substrates. The influence of factor XIIIa-induced cross-linking of fibrin was not examined. It is concluded from these findings that fibrinogen and non-cross-linked fibrin I are equally good substrates for plasmin cleavage of the NH₂-terminal end of the Bβ chain.     

Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood--different effects of collagen,TRAP and calcium ionophore A23187

We have studied the effects of different platelet agonists on phosphatidylserine (PS) exposure and clotting times in blood without anticoagulants. Similar reductions in clotting time were obtained for collagen, TRAP-6 or calcium ionophore A23187 (50 micro mol/L), in spite of huge differences in PS expression [6.7 +/- 2.4%, 2.3 +/- 0.5% and 99.9 +/- 0.1%, respectively (mean +/- SD, n = 5)]. Furthermore, the clotting times were much longer for samples with A23187 exposing the same amounts of PS as samples with collagen or TRAP-6. Annexin V reversed the clotting time reduction, but could not prevent coagulation. Addition of phospholipid vesicles containing 20% PS neither affected the clotting times nor induced clotting in recalcified, platelet-free plasma. We conclude that platelet PS exposure is necessary, but not sufficient, for the coagulation amplification observed when platelets are stimulated via physiological receptors in a whole blood environment.