Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.     

Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor of reaction mechanism

Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor.enzyme template bridging by heparin. Rationales behind this novel concept are discussed.     

Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition

Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.     

Allosteric activation of antithrombin critically depends upon hinge region extension

Antithrombin (AT) inhibits most of the serine proteases generated in the blood coagulation cascade, but its principal targets are factors IXa, Xa, and thrombin. Heparin binding to AT, via a specific pentasaccharide sequence, alters the conformation of AT in a way that promotes efficient inhibition of factors IXa and Xa, but not of thrombin. The conformational change most likely to be relevant to protease recognition is the expulsion of the N-terminal portion of the reactive center loop (hinge region) from the main beta-sheet A. Here we investigate the hypothesis that the exosites on the surface of AT are accessible for interaction with a protease only when the hinge region is fully extended, as seen in the related Michaelis complex between heparin cofactor II and thrombin. We engineered a disulfide bond between residues 222 on strand 3A and 381 in the reactive center loop to prevent the extension of the hinge region upon pentasaccharide binding. The disulfide bond did not significantly alter the ability of the variant to bind to heparin or to inhibit thrombin. Although the basal rate of factor Xa inhibition was not affected, that of factor IXa inhibition was reduced to the limit of detection. In addition, the disulfide bond completely abrogated the pentasaccharide accelerated inhibition of factors Xa and IXa. We conclude that AT hinge region extension is the activating conformational change for inhibition of factors IXa and Xa, and propose models for the progressive and activated AT Michaelis complexes with thrombin, factor Xa, and factor IXa.     

Antithrombin in vertebrate species: conservation of the heparin-dependent anticoagulant mechanism

Heparin is thought to regulate the rate of mammalian blood clotting by enhancing the activity of antithrombin, an inhibitor of coagulation enzymes. The present study establishes that this same inhibitor is present in the blood plasma of each of the terrestrial vertebrate groups including mammals, birds, reptiles, and amphibians. In each case, an inhibitor with remarkably similar properties to human antithrombin was isolated by affinity chromatography on immobilized porcine heparin. The purified vertebrate inhibitors all show the following physical and functional homologies to human antithrombin: (i) heparin-enhanced inhibition of both bovine thrombin and human Factor Xa, (ii) molecular masses of approximately 60,000, and (iii) heparin-induced increases in ultraviolet fluorescence. Also, the heparin-binding interaction of vertebrate antithrombins is highly selective with each demonstrating the same rigid specificity for heparin species fractionated on the basis of their affinity for human antithrombin. This common ability of vertebrate antithrombins to discriminate among heparins is accomplished by a nearly unvarying equilibrium binding constant for the high-affinity heparin species. Thus, the present results suggest that the anticoagulant relationship of heparin and antithrombin was established at an early point in the evolution of the coagulation system and has been highly conserved since that time.     

Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin

'Thrombin aptamers' are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion-binding exosite-I. The effect of aptamer-thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased approximately two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII-heparin and HCII-dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII-glycosaminoglycans.     

Mutation study of antithrombin: the roles of disulfide bonds in intracellular accumulation and formation of russell body-like structures

Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds and a deficiency of it is associated with venous thrombosis. Recently, we prepared CHO cells overexpressing a novel mutant, AT(C95R), with a disulfide bond removed, and revealed that this mutant remained for a long time in the endoplasmic reticulum (ER) without being degraded and also accumulated in newly formed membrane structures that resembled Russell bodies (RB) [Tanaka, Y. et al. (2002) J. Biol. Chem. 277, 51058-51067]. In this study, we replaced each of the individual cysteine residues of AT with an arginine and also two paired cysteine residues with arginines. We stably expressed these mutant ATs in CHO cells, and examined the roles of each cysteine residue or disulfide bond in the accumulation of mutant ATs and the formation of RB-like structures. In pulse-chase experiments, the secretion of mutant ATs with single mutations decreased approximately 1/5-1/50 times compared to that of the wild type AT. All of the mutant ATs were retained in the ER and were also found to accumulate in the RB-like structures. On the other hand, the fates of mutant ATs with double mutations fell into two categories. Secretion of mutant AT(C8R,C128R) decreased only approximately 1/2 times and no RB-like structures appeared. Mutants AT(C21R,C95R) and AT(C247R,C430R) exhibited similar secretion kinetics to the mutant ATs with the single mutations and were found in RB-like structures. On a sucrose gradient, all of the mutant ATs that induced RB-like structures migrated as oligomeric structures, whereas wild type AT and AT(C8R,C128R) migrated as monomers. Further, to clarify the morphological pathway through which RB-like structures are formed, we prepared CHO cells in which the expression of AT(C95R) was controlled by the Tet-On system. During expression of AT(C95R), RB-like structures formed through expansion of the ER. These results suggest that the correct folding with each disulfide bond is essential for the secretion of AT and oligomerization of mutant ATs in the ER is involved in the formation of RB-like structures.     

Protease specificity and heparin binding and activation of recombinant protease nexin I

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.     

Predominant contribution of surface approximation to the mechanism of heparin acceleration of the antithrombin-thrombin reaction. Elucidation from salt concentration effects

Heparin has been shown to accelerate the inactivation of alpha-thrombin by antithrombin III (AT) by promoting the initial encounter of proteinase and inhibitor in a ternary thrombin-AT-heparin complex. The aim of the present work was to evaluate the relative contributions of an AT conformational change induced by heparin and of a thrombin-heparin interaction to the promotion by heparin of the thrombin-AT interaction in this ternary complex. This was achieved by comparing the ionic and nonionic contributions to the binary and ternary complex interactions involved in ternary complex assembly at pH 7.4, 25 degrees C, and 0.1-0.35 M NaCl. Equilibrium binding and kinetic studies of the binary complex interactions as a function of salt concentration indicated a similar large ionic component for thrombin-heparin and AT-heparin interactions, but a predominantly nonionic contribution to the thrombin-AT interaction. Stopped-flow kinetic studies of ternary complex formation under conditions where heparin was always saturated with AT demonstrated that the ternary complex was assembled primarily from free thrombin and AT-heparin binary complex at all salt concentrations. Moreover, the ternary complex interaction of thrombin with AT bound to heparin exhibited a substantial ionic component similar to that of the thrombin-heparin binary complex interaction. Comparison of the ionic and nonionic components of thrombin binary and ternary complex interactions indicated that: 1) additive contributions of ionic thrombin-heparin and nonionic thrombin-AT binary complex interactions completely accounted for the binding energy of the thrombin ternary complex interaction, and 2) the heparin-induced AT conformational change made a relatively insignificant contribution to this binding energy. The results thus suggest that heparin promotes the encounter of thrombin and AT primarily by approximating the proteinase and inhibitor on the polysaccharide surface. Evidence was further obtained for alternative modes of thrombin binding to the AT-heparin complex, either with or without the active site of the enzyme complexed with AT. This finding is consistent with the ternary complex encounter of thrombin and AT being mediated by thrombin binding to nonspecific heparin sites, followed by diffusion along the heparin surface to a unique site adjacent to the bound inhibitor.     

Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III.

The relationship between thrombomodulin-associated O-linked glycosammoglycans (GAGs) and the exogenous GAGs heparin or dermatan sulfate was studied in the inhibition of thrombin by antithrombin III (AT III) or heparin cofactor II (HC II). Both rabbit thrombomodulin (TM) and two glycoforms (a high-Mr form containing GAGs and a low-Mr form lacking the majority of O-linked GAGs) of a recombinant human TM deletion mutant (rec-TM) were used. The rapid inactivation of thrombin by HC II in the presence of dermatan sulfate was prevented by both the high-Mr rec-TM and the rabbit TM. In contrast, both rabbit TM treated with chondroitin ABC lyase to remove O-linked GAGs and the low-Mr form of rec-TM had only weak protecting effects. In the absence of exogeneous dermatan sulfate, thrombin inhibition by a high concentration of HC II was slightly accelerated by the high-Mr form of rec-TM but protected by rabbit TM. When thrombin inhibition by AT III in the presence of heparin was studied, both high-Mr rec-TM and rabbit TM again invoked a similar reduction of inactivation rates, whereas in the absence of exogenous heparin, both high-Mr forms accelerated thrombin inhibition by AT III. The diverse reactivities of various forms of TM towards HC II and AT III were also observed during protein C activation by the thrombin-TM complex. These results suggest that thrombin activity at the vessel wall or in fluid phase may undergo major kinetic modulations depending on the type of protease inhibitor, the presence or absence of exogenous GAGs and the glycosylation phenotype of TM. The dependence of TM anticoagulant function on the presence of an intrinsic GAG moiety suggests that variant glycoforms of this endothelial cell cofactor may be expressed differently in a species-, organ-, or tissue-specific manner as a means to regulate TM function in diverse vasculatures.     

Antithrombin-mediated anticoagulant activity of sulfated polysaccharides: different mechanisms for heparin and sulfated galactans

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.     

Anticoagulant and anticomplement effects of an endogenous inhibitor from hepatopancreas of red king crab (<Emphasis Type="Italic">Paralithosed camtschaticus</Emphasis>) on human blood

Serpins are the superfamily of serine and cysteine protease inhibitors (SERine Protease Inhibitors) acting by an irreversible suicide mechanism. A novel serpin from hepatopancreas of red king crab (Paralithosed camtschaticus) was isolated and its effect on the process of human blood plasma coagulation was investigated. The investigated serpin exhibited a significant anticoagulant effect, which dramatically increased in the combination with heparin. The study of the crab serpin on C1s (C1 esterase) revealed its competition with the C1 inhibitor from blood plasma. Although the inhibitor weakly influenced thrombin activity, inhibition constant for C1s was (2.02 ± 0.71) 10⁻⁷ M. Unlike the C1 inhibitor the novel red king crab serpin does not inhibit fibrinolysis but inhibits blood coagulation. This creates certain clinical perspectives.     

Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin

Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.     

Control of the coagulation system by serpins. Getting by with a little help from glycosaminoglycans

Members of the serine protease inhibitor (serpin) superfamily play important roles in the inhibition of serine proteases involved in complex systems. This is evident in the regulation of coagulation serine proteases, especially the central enzyme in this system, thrombin. This review focuses on three serpins which are known to be key players in the regulation of thrombin: antithrombin and heparin cofactor II, which inhibit thrombin in its procoagulant role, and protein C inhibitor, which primarily inhibits the thrombin/thrombomodulin complex, where thrombin plays an anticoagulant role. Several structures have been published in the past few years which have given great insight into the mechanism of action of these serpins and have significantly added to a wealth of biochemical and biophysical studies carried out previously. A major feature of these serpins is that they are under the control of glycosaminoglycans, which play a key role in accelerating and localizing their action. While further work is clearly required to understand the mechanism of action of the glycosaminoglycans, the biological mechanisms whereby cognate glycosaminoglycans for each serpin come into contact with the inhibitors also requires much further work in this important field.     

The role of heparin in the thrombin-antithrombin III reaction

The effect of heparin on the kinetics of inactivation of thrombin by antithrombin III (AT) has been investigated in order to distinguish between two possible mechanisms. Either (1) heparin activates AT to make it a (kinetically) more effective inhibitor, or (2) heparin makes thrombin more susceptible to inhibition by AT. The results were consistent only with mechanism 1. The experimental approach was to premix heparin with either thrombin or AT and then to measure the rate of association of the two proteins in the rapid-mixing stop-flow spectrophotometer. Reactions were followed spectrophotometrically by observing displacement of the dye proflavine from the active site of thrombin as AT binds. Only premixing AT with heparin accelerated the reaction compared to control (no heparin); the observed second-order rate constant was enhanced by a factor of 200–400. Premixing of thrombin with heparin was without effect on the rate of association with AT. If heparin was premixed with both proteins before reaction, the rate was as slow as the control, indicating that heparin bound to thrombin is actually inhibitory to the association of enzyme with activated AT.     

Residues Tyr253 and Glu255 in strand 3 of beta-sheet C of antithrombin are key determinants of an exosite made accessible by heparin activation to promote rapid inhibition of factors Xa and IXa

We previously showed that conformational activation of the anticoagulant serpin, antithrombin, by heparin generates new exosites in strand 3 of beta-sheet C, which promote the reaction of the inhibitor with the target proteases, factor Xa and factor IXa. To determine which residues comprise the exosites, we mutated strand 3C residues that are conserved in all vertebrate antithrombins. Combined mutations of the three conserved surface-accessible residues, Tyr253,Glu255, and Lys257, or of just Tyr253 and Glu255, but not any of these residues alone, was sufficient to reproduce the exosite defects of a strand 3C antithrombin-alpha1-proteinase inhibitor chimera in reactions of the heparin-activated variants with both factor Xa and factor IXa. Importantly, the exosite-defective antithrombins bound heparin with nearly wild-type affinities, and the heparin-activated mutants showed near normal reactivities with thrombin, a protease that does not utilize the exosite. Mutation of the conserved but partially buried strand 3C residue, Gln254, the reactive loop P6' residue, Arg399, which interacts with Glu255, or a residue proposed to constitute the exosite from modeling studies, Glu237, all produced minimal effects on antithrombin reactivity with thrombin, factor Xa, and factor IXa in the absence or presence of heparin. Together, these results indicate that Tyr253 and Glu255 are key exosite determinants responsible for promoting the reactions of conformationally activated antithrombin with both factor Xa and factor IXa.     

Evidence for essential lysines in heparin cofactor II

Covalent modification with pyridoxal 5'-phosphate was used to study the function of lysyl residues in heparin cofactor II, a heparin-dependent plasma protease inhibitor. Reduction of the Schiff base with sodium borohydride resulted in modification of 3-4 lysyl residues of heparin cofactor II at high concentrations of pyridoxal 5'-phosphate, one of which was protected in the presence of heparin. The antithrombin activity of modified heparin cofactor II was enhanced compared to the native protein. However, the heparin cofactor activity for thrombin inhibition was reduced significantly or completely eliminated in the modified protease inhibitor depending on the extent of phosphopyridoxylation. In contrast to native heparin cofactor II, the modified protease inhibitor did not bind to a heparin-agarose column. The results suggest that lysyl residues are essential for heparin cofactor activity during thrombin inhibition.     

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin''s active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.     

Interaction of pregnancy-associated plasma protein-A (PAPP-A) with coagulation: a bioassay for PAPP-A

The observation that pregnancy-associated plasma protein A (PAPP-A) concentrations are higher in plasma compared to serum obtained from the same patient, together with fact that PAPP-A binds to heparin, prompted us to study the interaction between PAPP-A and the clotting system. It was determined that pure PAPP-A inhibits thrombin-induced coagulation of citrated plasma. The presence of antithrombin III (AT III) was necessary since PAPP-A had no inhibitory effect on coagulation of AT III-depleted plasma. The effect of PAPP-A is thus similar to that of heparin. This property of PAPP-A was used to develop a bioassay. Thrombin-induced polymerization of purified fibrinogen was measured in a spectrophotometer. AT III is a weak inhibitor of polymerization, but its effect is magnified in the presence of PAPP-A or heparin. The residual thrombin activity, when plotted against the concentration of PAPP-A, gives a linear relationship. The assay conditions developed allow maximal sensitivity and reproducibility. The kinetics of inhibition due to PAPP-A and heparin was first order. With this bioassay, activities of PAPP-A molecules isolated by the same technique from different fetomaternal compartments were compared.