Differential impact of conventional and low-dose oral hormone therapy (HT), tibolone and raloxifene on functionality of the activated protein C system.

Recent studies have shown that hormone therapy (HT) is associated with an acquired resistance to activated protein C (APC). The aims of the present study were to evaluate a possible dose-response relationship and differential effects of different HT regimens on functionality of the APC system. Two hundred two healthy women were randomly assigned to receive treatment for 12 weeks with tablets containing either low-dose HT containing 1 mg 17ss-oestradiol + 0.5 mg norethisterone acetate (NETA) (n = 50), conventional-dose HT containing 2 mg 17ss-oestradiol and 1 mg NETA (n = 50), 2.5 mg tibolone (n = 51), or 60 mg raloxifene (n = 51). Normalized APC system sensitivity ratios (nAPCsr) were determined in plasma collected at baseline and after 12 weeks using a thrombin generation-based APC resistance test probed with either recombinant APC (rAPC) or thrombomodulin (rTM). NAPCsr increased in both the conventional- and low-dose HT groups, consistent with reduced sensitivity to APC. The increase was slightly more pronounced in the conventional-dose group, but the difference between the two HT groups was not statistically significant. The sensitivity to APC was only marginally altered in those allocated to tibolone. Consequently, tibolone showed a different phenotype as compared with the low-dose HT group. A small increase in nAPCsr with both rAPC and rTM was seen in the raloxifene-group, but the increase was less than in the low-dose HT group. Our findings indicate that oestrogen-progestin therapy induces an APC resistant phenotype, which may be related to dose, whereas tibolone and raloxifene only marginally alter the sensitivity to APC.

Randomized, placebo-controlled trial of low molecular weight heparin in active ulcerative colitis.

BACKGROUND: In several open and 1 controlled trial, unfractionated heparin was effective in the treatment of active ulcerative colitis (UC). Low molecular weight heparin (LMWH) had a similar effect in several open studies. METHODS: We studied the efficacy, safety, and tolerability of LMWH in mild to moderately active UC in a randomized, double-blind, placebo-controlled trial. In all, 29 patients with a mild or moderate recurrence of UC during salicylate treatment were randomized to receive either reviparin 3,436 IU (n = 15) subcutaneously twice daily or placebo (n = 14). The study period was 8 weeks. Treatment was discontinued if there was no improvement at 4 weeks or at any disease progression. Primary outcome measure was clinical improvement at 8 weeks measured by the Colitis Activity Index (CAI) and the Clinical Symptoms Grading (CSG, based on the CAI). Endoscopic and histologic grading and quality of life as measured by the Inflammatory Bowel Disease Questionnaire (IBDQ) were secondary outcome measures. Patients were closely monitored for adverse events. RESULTS: Twenty of 29 patients finished the 8-week treatment period (reviparin versus placebo: 11 versus 9; P = 0.70). There was no difference in CSG, CAI, endoscopic and histologic grading, or IBDQ. Treatment was well tolerated and no serious adverse events occurred. CONCLUSION: In this study, treatment with LMWH showed no significant clinical advantage compared to placebo in mild to moderately active UC.

Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule.

Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.

Autocatalytic peptide bond cleavages in prothrombin and meizothrombin.

During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg51-Thr52/Arg54-Asp55 in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s-1), Arg155-Ser156 in prothrombin (k = 2 x 10(4) M-1 s-1), and Arg284-Thr285 in prethrombin 1 (k = 0.02 x 10(4) M-1 s-1). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg51-Thr52/Arg54-Asp55) was not detectable, whereas cleavage at Arg155-Ser156 (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg284-Thr285 (formation of prethrombin 2 des-1-13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t1/2 = approximately 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 microM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s-1 in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg284-Thr285 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des-1-13 by a combination of intramolecular (k = 0.8 x 10(-4) s-1) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s-1). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s-1), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s-1) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) M-1 s-1, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.

The Ca2+-mobilizing potency of alpha-thrombin and thrombin-receptor-activating peptide on human platelets -- concentration and time effects of thrombin-induced Ca2+ signaling.

In single platelets and in suspensions of platelets, alpha-thrombin evokes dose-dependent, transient increases in cytosolic Ca2+ concentration, [Ca2+]i, which are more prolonged than the [Ca2+]i transients evoked by other platelet agonists such as the thrombin-receptor-activating hexapeptide SFLLRN, thromboxane A2 analog U46619, and ADP. As a quantity taking into account both the magnitude and length of the Ca2+ response, we defined the Ca2+-mobilizing potency (CMP) of an agonist as the integrated rise in [Ca2+]i during the time of the Ca2+ signal. It was observed that: (a) the CMP increased with the agonist concentration in a saturating way, its maximal value being about four-times higher with alpha-thrombin than with SFLLRN; (b) the high CMP of alpha-thrombin was for only a small part due to endogenous production of ADP or thromboxane, and was mainly a consequence of prolonged influx of external Ca2+; (c) the CMP declined when alpha-thrombin was inactivated during the course of the Ca2+ signal; (d) CMP values increased with the agonist concentration upon sequential addition of increasing amounts of alpha-thrombin or SFLLRN; (e) when alpha-thrombin was gradually added to the platelets or formed by an in situ reconstituted prothrombinase system (with factor Xa, factor Va, and prothrombin), integrated Ca2+ responses were a function of the product of the alpha-thrombin concentration and the time of its presence. However, in these cases, the final CMP values were independent of the rate of alpha-thrombin addition or formation. We conclude that alpha-thrombin-induced Ca2+ signals in platelets rely largely upon Ca2+ influx, are not, or only slightly, subjected to homologous desensitization, and reflect the enzymatic capacity of alpha-thrombin to cleave protease-activated receptors. Thus, the high and prolonged Ca2+ signal induced by alpha-thrombin is due to continuous receptor cleavage without desensitizing effects of previously cleaved receptors.

Synthesis of peptide p-nitroanilides mimicking fibrinogen- and hirudin-binding to thrombin. Design of slow reacting thrombin substrates.

The design and synthesis of 20 peptide p-nitroanilides is described. The nitroanilides are used as thrombin substrates that share uncommon properties. These substrates are tested for their applicability to measure thrombin generation in activated plasma. This technique requires substrates that must slowly but selectively be hydrolyzed by thrombin. To ensure selectivity, thrombin's natural substrate and its most potent inhibitor were used as lead compounds. Eighteen peptide p-nitroanilides were synthesized using human fibrinogen A alpha [7-16] decapeptide as lead structure since this fragment constitutes the minimal sequence which binds to thrombin with high affinity. Two other chromogenic substrates were designed using 5-amino-2-nitrobenzoic acid as the chromophore. A peptide giving subsite interactions with the fibrinogen recognition exosite was coupled to the carboxylic function. As the peptide segment, the carboxyl terminal part of hirudin (residues 50-65) was chosen to ensure highly specific thrombin recognition. From the 20 nitroanilides synthesized, we indeed obtained a number of compounds which can be used as substrate in the thrombin generation assay.

Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506-->Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (< 5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 x 10(7) M-1s-1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 x 10(6) M-1s-1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 x 10(6) M-1s-1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s-1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km = 196 nM and kcat = 0.37 s-1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus).

From the saliva of the vampire bat Desmodus rotundus, we isolated an unknown anticoagulant protein which we have named draculin. Its molecular mass as determined by non-reduced SDS-PAGE is about 83 kDa. The reduced polypeptide shows a slower migration. HPLC in a molecular sieve matrix yields a single, symmetrical peak corresponding to 88.5 kDa. Isoelectric focusing shows an acidic protein with pI = 4.1-4.2. Aminoacid analysis is compatible with a single chain polypeptide of about 80 kDa. Cyanogen bromide cleavage yields a single 16-aminoacid peptide, corresponding to the amino-terminus of the native molecule. Draculin inhibits the activated form of coagulation factors IX and X. It does not act on thrombin, trypsin, chymotrypsin and does not express fibrinolytic activity. The inhibition is immediate and not readily reversible, with a stoichiometry of about two molecules of draculin per molecule of factor IXa or Xa. Surprisingly, the inhibitory activity against either factor is not affected by the presence of the other. Draculin binds quantitatively to either immobilised factor Xa or factor IXa. Our preliminary interpretation is that there are two forms of draculin that hardly differ in structure. Both bind to factor Xa and to factor IXa but one form inhibits factor Xa and the other inhibits factor IXa. When added to plasma, draculin increases the lag phase as well as the height of the peak of thrombin generation.

Annexin V inhibits the procoagulant activity of matrices of TNF-stimulated endothelium under blood flow conditions.

A human ex vivo thrombosis model was used to investigate whether recombinant annexin V (rANV) can prevent thrombus formation under venous and arterial blood flow conditions. In this model, blood from an antecubital vein of healthy donors was allowed to flow directly over the extracellular matrix of tumor necrosis factor-stimulated endothelial cells (TNF-ECMs). TNF-ECMs were preincubated with rANV (2.9 mumol/L) for 30 minutes. With this rANV concentration all binding sites present on TNF-ECMs (1.6 +/- 0.5 x 10(12)/cm2) are occupied, and a maximal inhibition was observed in a tissue factor-dependent clotting assay. Fibrin deposition and platelet and leukocyte adhesion were measured on the rANV-treated and nontreated TNF-ECMs. Nontreated TNF-ECMs were used as controls. rANV inhibited fibrin deposition by 81% at a wall shear rate of 100 s-1. A nonsignificant inhibition was also observed at 650 s-1. Platelet-matrix adhesion, which is more prominent at higher shear rates, was significantly decreased by 60% at 100 s-1 but not at 650 s-1. The average leukocyte adherence was nonsignificantly lowered at 100 s-1. Virtually no leukocytes adhered at 650 s-1. The results demonstrated that rANV can inhibit blood coagulation under venous blood flow conditions and may serve as an antithrombotic drug.

Activation of factor X and its regulation by tissue factor pathway inhibitor in small-diameter capillaries lined with human endothelial cells.

The activation of factor X at the surface of endothelial cells was investigated under controlled flow conditions. A method is described for preparing polyethylene capillaries whose inner walls are covered with a confluent layer of human umbilical vein endothelial cells. To obtain a stable and unperturbed layer of endothelial cells it was essential to pre-perfuse the endothelialized capillaries with medium for about 18 hours. At this stage no tissue factor activity could be detected, but when the seeded cells were perfused with medium containing tumor necrosis factor (TNF) a maximum steady-state rate of factor Xa production (16 fmol factor Xa/min/cm2) was observed within 8 hours. Further experiments were performed with endothelial cells incubated for 4 hours with TNF. Factor Xa was produced at a rate of 7 fmol factor Xa/min/cm2 on perfusion of the capillaries with factor X (100 nmol/L) and factor VII (0.1 U/mL) at a shear rate of 34 s-1. The extracellular matrix preparations of these cells produced factor Xa at a 20-fold higher rate (150 fmol factor Xa/min/cm2). In both cases factor Xa formation was dependent on the presence of factor VII and was completely inhibited when the perfusate also contained 5 nmol/L recombinant tissue factor pathway inhibitor (rTFPI). Pre-perfusion with factor Xa-TFPI complex in the absence of factor VIIa caused a much lesser inhibitory effect, suggesting that TFPI-mediated neutralization of endothelial cell and matrix tissue factor activity requires the presence of factor VIIa in addition to the presence of factor Xa.

Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor.

The binding of affinity-purified anticardiolipin antibodies (ACA) to liposomes that contained cardiolipin or phosphatidylserine was investigated. ACA bound to these liposomes only in the presence of plasma or serum, which indicated a requirement for a plasma component. This component--referred to as aca-cofactor--was purified; its activity to support ACA binding to liposomes that contained cardiolipin was not destroyed by heat (10 min at 90 degrees C), but was greatly diminished on incubation with trypsin. aca-cofactor bound liposomes that contained negatively charged phospholipid but had no affinity for liposomes that contained neutral phospholipid (eg, phosphatidylcholine); this binding was independent of calcium ions. aca-cofactor was essential for ACA to bind to liposomes that contained cardiolipin or phosphatidylserine and, when coated on a microtitre plate in the absence of any phospholipid, aca-cofactor was an apparent antigen for ACA in an enzyme-linked immunosorbent assay. aca-cofactor is a single chain polypeptide with an apparent molecular weight of 50 kD (non-reduced), which increases to 70 kD upon reduction, and its properties closely resemble those of beta 2-glycoprotein I (apolipoprotein H).

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.

Localization of the inhibitory site(s) of pentosan polysulphate in blood coagulation.

We studied the inhibitory effect of pentosan polysulphate (PPS, Hémoclar) on thrombin formation in blood coagulation. In contrast to a current hypothesis the antithrombin III independent effect of PPS on blood coagulation is not caused by preventing the binding of the factors IX, IXa, X, Xa, VIII, V, Va and II onto procoagulant phospholipids. We investigated the activation by thrombin of factors I, V and VIII. A strong inhibitory effect of PPS on factor VIII activation could be observed. Inhibition of the activation of factor V to the same extent requires about 30-fold higher concentrations of PPS, whereas the activation (clotting) of fibrinogen is not inhibited. The effect of PPS on factor VIIIa is two-fold: A) it inhibits its formation and B) it inhibits its function probably by the formation of a factor VIIIa-PPS complex. Prothrombinase, constituted of purified factors Xa, Va and phospholipids was not inhibited by PPS, neither were incomplete forms of this enzyme, lacking phospholipids or factor Va. The complete factor X activating enzyme (factors IXa, VIIIa and phospholipids), however, was strongly inhibited, but incomplete forms, lacking factor VIII, were not. The inhibition of the complete enzyme can be explained by reversible binding of PPS to factor VIIIa (causing an inhibition of its function) and it is not an effect on the enzymatic function of the complete enzyme. On saturation of the enzyme with an excess of factor VIIIa no inhibition by PPS is noticed. We postulate therefore that the antithrombin III independent inhibitory effect of PPS on thrombin generation on blood coagulation is by interaction with factor VIIIa. This effect is additional to the heparin-like action of PPS, i.e. potentiation of the activity of antithrombin III and/or heparin cofactor II.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a novel protein from bovine aorta that inhibits coagulation. Inhibition of the phospholipid-dependent factor-Xa-catalyzed prothrombin activation, through a high-affinity binding of the anticoagulant to the phospholipids.

A novel inhibitor of blood coagulation has been isolated from the intima of bovine aorta. The inhibitor, vascular anticoagulant (VAC), has been purified to an active fraction that contains two Coomassie-blue-staining bands (Mr = 34,000 and Mr = 32,000, as judged by sodium dodecyl sulfate/polyacrylamide electrophoresis). Both bands are single-chain proteins, having no glycoprotein features. Furthermore, they do not contain any detectable 4-carboxyglutamic acid residues. Both proteins have an identical isoelectric pH of approximately 4.5. VAC binds in the presence of calcium ions to a bilayer consisting of 20% dioleoylglycerophosphoserine and 80% dioleoylglycerophosphocholine with a Kd = 6 nM. The binding is dependent on the calcium concentration: half-saturation of binding occurs at a calcium concentration of 0.8 mM. The binding is completely reversible with EDTA. Furthermore the phospholipid/VAC ratio at saturation was n = 112 and n = 32 mol/mol for 0.5 mM Ca2+ and 2 mM Ca2+, respectively. Binding does not occur between VAC and pure dioleoylglycerophosphocholine. In a system with purified coagulation factors VAC inhibits the activation of prothrombin by factor Xa and calcium only in the presence of negatively charged phospholipids. VAC decreases the Vmax and increases the Km of the factor-Xa-catalyzed prothrombin activation. Based on these results, we conclude that we have purified from bovine aortic intima an anticoagulant protein, which exerts its activity through a calcium-dependent binding to negatively charged phospholipids, and thus interferes with the assembly of prothrombinase on the phospholipid surface.

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord.

An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide gel electrophoresis and inactivation studies using proteolytic enzymes indicate that the anticoagulant activity is associated with a polypeptide with an apparent Mr of 32 000. The anticoagulant inhibits thromboplastin as well as factor Xa induced clotting but does not affect thrombin initiated fibrin formation. The anticoagulant inhibits the activation of prothrombin by the complete prothrombinase complex, by phospholipid bound factor Xa but not by free factor Xa. The inhibition is instantaneous and independent of the incubation time over the whole range of concentrations tested. Therefore, the anticoagulant is unlikely to be a phospholipase or a protease. Its action does not resemble that of the plasma protease inhibitors, but it probably interferes with the phospholipid--clotting factor interactions.

Comparison between the effect of pentosan polysulphate heparin and antithrombin III injections in antithrombin III deficient patients.

Pentosan polysulphate is an heparin analogue which acts via an antithrombin III (AT III) independent pathway. We compared the effect of this drug to that of heparin and AT III infusions in AT III deficient patients. Four patients with AT III congenital deficiency received on three different occasions: (i) an infusion of human AT III concentrate (20 U/kg or 40 U/kg), (ii) an intramuscular injection of pentosan polysulphate (2 mg/kg), (iii) a subcutaneous calcium heparin injection (100 U/kg). AT III infusion inhibits the excessive thrombin generation (46% of inhibition) observed in the plasma of AT III deficient patients during at least 12 hours, but does not modify the factor Xa formation. On the contrary, pentosan polysulphate has a marked effect on both thrombin (62% of inhibition) and factor Xa generation (57% of inhibition) still present 8 hours after injection. Heparin injection has the same effect, more prolonged, as pentosan polysulphate on thrombin generation but is not so effective on impairing factor Xa generation (27% of inhibition). The marked effect of pentosan polysulphate on thrombin and factor Xa generation in these patients is due to its AT III independent mechanism of action.

Measurement of macrophage cellular procoagulant activity.

We developed a simple technique for the measurement of the procoagulant activity exposed on the surface of macrophages. The cells are isolated, adhered to plastic surfaces, and assayed in the same device. This approach allows us to study the microcoagulation on the surface of intact macrophages by sensitive and specific clotting tests.

Clotting factors secreted by monocytes and macrophages: analytical considerations.

The secretion of clotting factors by rat spleen macrophages and human peripheral blood monocytes has been studied. The results show that the amount of clotting factors measured depends critically upon the characteristics of the assay system used. The presence of warfarin, salicylic acid or thrombin in the culture medium is shown to decrease the vitamin K dependent clotting factor activity in the supernatant after in vitro culture of rat spleen macrophages and human peripheral blood monocytes.

The inhibition of platelet prothrombinase activity by prostacyclin.

Prostacyclin is able to inhibit the development of platelet prothrombinase activity. This inhibition, which also occurs with dibutyryl cAMP, is presumably due to the ability of prostacyclin to prevent the formation of a negatively charged phospholipid surface at the exterior half of the platelet membrane. Generation of this procoagulant surface, as induced by platelet activation with collagen plus thrombin, does not depend on thromboxane A2 formation.

Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition.

The interaction between factor Xa and factor Va was investigated both in solution and in the presence of phospholipid vesicles with varying contents of phosphatidylserine. The binding parameters were inferred from the kinetics of prothrombin activation. Factor Xa and factor Va form in solution an equimolar complex with a dissociation constant of 3.3 X 10(-9) M. Phospholipid vesicles promote the formation of the factor Xa-Va complex. The Kd of complex formation is dependent on both the phospholipid concentration and the composition of the phospholipid vesicle. For the interaction between factor Xa and factor Va in the presence of phospholipid vesicles containing 40 mol % dioleoylphosphatidylserine (DOPS) and 60 mol % dioleoylphosphatidylcholine (DOPC), the Kd increases linearly with increasing phospholipid concentration. In the presence of 10 microM phospholipid (DOPS/DOPC, 40/60 mol/mol) Kd = 3 X 10(-11) M. When the mole percentage of DOPS in the phospholipid vesicles is lowered from 20 to 5 mol %, there is a gradual increase of the Kd. In the presence of 10 microM phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC Kd = 2.8 X 10(-10) M. The Kd measured in the presence of phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC is independent of the phospholipid concentration. Two models are discussed that can quantitatively explain the effect of phospholipid vesicles on the complex formation between factor Xa and factor Va. Studies on the effect of the polypeptides with Mr 80 000 and Mr 94000 of which factor Va is composed on the Kd of the factor Xa-Va complex suggest that factor Xa binding to factor Va requires a Ca2+-mediated interaction between the two polypeptides.