dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide Research Articles

Distinct Mechanism of Anti-Hemophilic FVIII Activation By Nascent FXa in the Tissue Factor-FVIIa Coagulation Initiation Complex Resistant to FXa-Directed Inhibition

Hemorrhagic complications may occur during anticoagulant therapy, but clinical studies indicate that FXa-directed anticoagulants are not inferior to vitamin K antagonists in preventing thrombosis while decreasing the incidence of intracranial bleeding. Anti-hemophilic FVIII has a central role in hemostasis, thus we hypothesized that elucidating effects of FXa-directed anticoagulants on FVIII function could advance understanding of mechanisms differentiating hemostasis and thrombosis. Currently, FVIII is thought to be activated by limited amounts of thrombin (FIIa) initially produced by FXa generated by the tissue factor (TF)-FVIIa complex. Activated FVIII (FVIIIa) then amplifies FXa generation through intrinsic tenase (FVIIIa-FIXa) complex, leading to more FIIa production. We now propose an alternative mechanism of FVIII activation resistant to the TF pathway inhibitor (TFPI) and direct anti-FXa anticoagulants. In this process, nascent FXa generated by the TF-FVIIa complex generates FVIIIa independently of FIIa feedback activation. We first verified whether TF-dependent FVIII activation by FXa can promote FIIa generation by FIXa-FVIIIa in human platelet-rich plasma (PRP). Addition of relipidated TF (0.15 pM) into PRP synergistically enhanced FIIa production by FIXa (20 pM) in a process requiring both FVIIa and FVIII. Synergistic FIIa generation was blocked by an anti-TF monoclonal antibody (MoAb) and the blockade was reversed by FVIIIa, supporting the concept that FVIII activation is a key TF-dependent function in the process. We further tested the hypothesis in vivo using a ferric chloride-induced mouse thrombosis model. Concurrent administration of anti-FXI and anti-TF MoAbs at individually ineffective doses markedly reduced fibrin deposition onto the lesion, suggesting that TF and contact phase (CP)-initiated pathways cooperate in generating coagulation proteases leading to thrombosis. Infusion of FVIIIa, but not FVIII, by-passed the inhibitory effect of the anti-TF MoAb, but full FXI inhibition still prevented thrombosis, in agreement with the concept FVIII activation in vivo is a TF-dependent function. We next evaluated whether the TF pathway can generate FVIIIa in reactions with purified components including FVIII, FX, FV and FII in which relipidated TF (50 pM) provided a limiting phospholipid surface. Addition of FIXa (10 nM) with FVIIa (200 pM) synergistically increased FXa generation even when the thrombin inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) was added or wild-type FII was replaced by the catalytically inactive FII S195A mutant. TF-FVIIa-induced FVIII activation was reduced, but not abolished, by DAPA. Thus, TF-FVIIa-mediated FVIII activation by FXa is sufficient for productive FVIIIa-FIXa complex assembly even without FIIa feedback activation. TFPI/protein S and antithrombin/pentasaccharide did not prevent FIIa-independent but inhibited FIIa-dependent FVIIIa generation, indicating that the newly discovered FIIa-independent functional link between TF and intrinsic pathways escapes control by physiologic inhibitors. To verify that FVIII activation is a distinct function of FXa possibly still assembled within the TF-FVIIa complex, we showed that the nematode anticoagulant protein (NAP) c2-stabilized complex of FXa, TF and catalytically inactive FVIIa (S195A) - in which FXa is the only active protease - but not free FXa with NAPc2, generated FVIIIa even in the presence of physiologic TFPI inhibition. At therapeutic concentrations, the FXa-directed anticoagulants, rivaroxaban and apixaban, moderately inhibited FVIII activation by nascent FXa, while both blocked FVIII activation by free and RussellÕs viper venom-activated FXa, indicating resistance of nascent FXa-mediated FVIIIa generation also to pharmacologic FXa inhibition. Inefficient inhibition of FVIII activation by FXa anticoagulants allowed FVIIIa/FIXa-dependent thrombin production even under TFPI control. In conclusion, persistent FVIII activation preserving hemostasis could explain why the antithrombotic efficacy of FXa inhibitors is associated with a relatively low risk of bleeding complications. Our findings provide new perspectives for designing antithrombotic intervention with lesser anti-hemostatic effects as well as for personalized evaluation of bleeding risk during anticoagulant therapy. DisclosuresGale:University of California, San Diego: University of California, San Diego Patents & Royalties. Gruber:Aronora, Inc: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Petersen:NovoNordisk A/S: Employment.

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

Incorporation of Factor Va into Prothrombinase Is Required for Coordinated Cleavage of Prothrombin by Factor Xa

Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320. Factor Va, along with phospholipid and Ca2+, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg320. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor VaFF/MI). The present studies were undertaken to determine whether loss of activity can be attributed to selective loss of efficiency at one or both of the two prothrombin-activating cleavage sites. Kinetic constants for the overall activation of prothrombin by prothrombinase assembled with saturating concentrations of recombinant mutant factor Va were calculated, prothrombin activation was assessed by SDS-PAGE, and rate constants for both cleavages were analyzed from the time course of the concentration of meizothrombin. Prothrombinase assembled with factor VaFF/MI had decreased k(cat) for prothrombin activation with Km remaining unaffected. Prothrombinase assembled with saturating concentrations of factor VaFF/MI showed significantly lower rate for cleavage of plasma-derived prothrombin at Arg320 than prothrombinase assembled with saturating concentrations of wild type factor Va. These results were corroborated by analysis of cleavage of recombinant prothrombin mutants rMz-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A), which can be cleaved only at Arg320 or Arg271, respectively. Time courses of these mutants indicated that mutations in the factor Xa binding site of factor Va reduce rates for both bonds. These data indicate that the interaction of factor Xa with the heavy chain of factor Va strongly influences the catalytic activity of the enzyme resulting in increased rates for both prothrombin-activating cleavages.

Ratcheting between Two Distinct Conformations of Substrate Drives the Sequential Cleavage of Prothrombin by Prothrombinase.

The conversion of human prothrombin to thrombin requires the cleavage of two peptide bonds. When catalyzed by prothrombinase, the reaction proceeds almost exclusively via initial cleavage at R320 followed by cleavage at R271 yielding meizothrombin (mIIa) as an intermediate. The scissile bonds are expected to be ~36 A apart in the zymogen. Yet, remarkably, evidence indicates that cleavage at these two sites is accomplished by a single type of exosite interaction that tethers the substrate to prothrombinase. The ability of prothrombinase to act on these spatially distinct sites, with such constraints, can be explained by a conformational change in the substrate following initial cleavage at R320. Cleavage at this site leads to internal salt bridge formation and a conformational transition to the proteinase. The role of the zymogen to proteinase transition in substrate cleavage was investigated by the use of a fully-carboxylated recombinant prothrombin derivative (IITAT) with the I-V-E sequence following R320 replaced with T-A-T. Thrombin produced by cleavage of IITAT exhibited ~0.2% of the catalytic activity observed with thrombin produced from wild type recombinant prothrombin (IIWT). IITAT can be cleaved at the R320 site but fails to undergo all subsequent conformational changes required for proteinase formation. SDS-PAGE and quantitative densitometry revealed that the action of prothrombinase on either IIWT or IITAT was consistent with ordered cleavage at R320 followed by cleavage at R271. The rates of consumption of IIWT and IITAT resulting from cleavage at R320 were equivalent. Cleavage at R320 in IITAT by prothrombinase is not detectably affected by the substituted P1′-P3′ sequence. The disappearance of IIWT was accompanied by a transient accumulation in mIIa that decreased to zero within 4 minutes while thrombin accumulated following a short delay. With IITAT, mIIa accumulated to higher levels and persisted for 45 minutes. Thrombin was produced with a lower rate and a longer delay phase. The findings imply a substantial decrease in the rate of the second cleavage reaction at R271 resulting from distant effects of the new P1′-P3′ residues following R320. The thrombin inhibitor, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA) had no effect on the cleavage reactions in IIWT. However, increasing concentrations of DAPA as high as 400 μM were found to systematically enhance the rate of thrombin formation from IITAT by correcting defective cleavage at R271. The data are consistent with the interpretation that IITAT can be cleaved normally at R320 but subsequent accessibility and cleavage at R271 is reduced because of a defect in internal salt bridge formation and the conformational change associated with proteinase formation. Rescue of this defect by high concentrations of DAPA likely relates to the stabilization of a proteinase-like conformation in the intermediate produced by cleavage of IITAT at R320. Our findings suggest an important role for the zymogen to proteinase transition in determining the sequential action of prothrombinase on the two sites in prothrombin. We propose that exosite-dependent tethering of two distinct conformations of the substrate to prothrombinase drives the presentation of distantly positioned cleavage sites to the active site of the enzyme and accounts for the sequential cleavage of prothrombin.

Aggregation of human platelets in plasma by porcine blood cells in vitro is probably mediated by thrombin generation.

The infusion of pig progenitor cells into baboons is associated with a thrombotic microangiopathy probably related to the interaction of these cells with the baboon endothelial cells and platelets. We have shown previously that pig peripheral blood mononuclear cells (p-PBMC), are able to activate the human coagulation cascade as they are able to generate thrombin when added to defibrinated plasma. In this work, we have tested the interaction of p-PBMC with human platelets to assess the capacity of p-PBMC to cause platelet aggregation and the possible role of complement activation in this aggregation. Human platelet aggregation assays, using collagen (1 or 2 microg/ml), were performed with platelets in platelet-rich plasma (PRP) or platelets washed by filtration. PRP or washed platelets were also incubated with p-PBMC or human PBMC (h-PBMC) at several concentrations and aggregation was measured. The effect of Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an inhibitor of thrombin, was studied on platelet aggregation caused by the pig cells. Complement activation was measured by deposition of fragment c derived from C3 splitting (C3c) on pig cells incubated with citrated platelet poor plasma (PPP). When human PRP was incubated with p-PBMC, aggregation was a consistent event quantitatively similar to that induced by collagen. No aggregation of washed platelets was observed when these were incubated with p-PBMC or h-PBMC. Aggregation of human platelets in PRP, induced by p-PBMC, was inhibited when DAPA (100 microm) was added to the incubation mixture (23%), indicating that the thrombin inhibitor blocked the capacity of p-PBMC to aggregate human platelets. No deposition of C3c fragments on p-PBMC was detected when the porcine cells were incubated for up to 20 min with citrated PPP. The fact is that p-PBMC induces human platelet aggregation in plasma being thrombin generation a likely explanation for this observation. Our data suggest that, in the system assayed, complement activation is not a cause of platelet aggregation. These findings are relevant for the clarification of the reported thrombotic microangiopathy complicating the intravenous infusion of pig cells in primates in attempts to induce pig tolerance in baboons.

α-thrombin and trypsin use different receptors to stimulate arachidonic acid metabolism

Rat liver cells (the C-9 cell line) are stimulated to metabolize arachidonic acid by α-thrombin, its receptor polypeptide, γ-thrombin, and trypsin. Prostaglandin (PG) I 2 synthesis stimulated by α-thrombin is inhibited by dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA), by hirudin, by the synthetic tyrosine-sulfated dodecapeptide corresponding to residues 53–64 of hirudin (hirugen), by the Tyr(SO 3H) 63-hirudin fragment 54–65 and by rabbit lung thrombomodulin. Stimulation of arachidonic acid metabolism by the receptor octapeptide, SFLLRNPN, is not affected by DAPA or hirudin. γ-Thrombin stimulates arachidonic acid metabolism but at 300 to 400-fold higher concentrations. Trypsin stimulates arachidonic acid metabolism. Trypsin's proteolytic activity is required—its ability to stimulate is abolished if it is incubated with Na-p-tosyl- l-lysine chloromethyl ketone (TLCK) or bovine pancreatic trypsin inhibitor. Prior treatment of the rat liver cells with α-thrombin blocks subsequent stimulation by α-thrombin, but not by trypsin, whereas prior treatment with trypsin blocks subsequent stimulation by trypsin, but not the activity stimulated by α-thrombin. Prior treatment of the cells with the serine-proteases, chymotrypsin, pancreatic or neutrophil elastase and thrombocytin from Bothrups atrox venom, block α-thrombin's activation of PGI 2 production, but not the activity stimulated by trypsin. These findings indicate that α-thrombin and trypsin stimulate PGI 2 production via different receptors.

Isolation and autolysis of human meizothrombin in the presence of dansylarginine-N-(3 -ethyl-1,5-pentanediyl)amide

Human meizothrombin, a transient intermediate in the activation of prothrombin to thrombin, was isolated in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA), an active site inhibitor of thrombin. Meizothrombin autolysis to meizothrombin autolysis to meizothrombin des fragment 1 (cleavage at Arg273- Thr274) at different DAPA concentrations was monitoed by gel electrophoresis or by the changed fluorescence intensity of the DAPA-protein complex. Meizothrombin autolysis could be described by apparent pseudo-first order kinetics and was not eliminated even in the presence of a 100-fold molar excess of DAPA. By fitting the autolysis rates observed at different DAPA concentrations to a simple inhibition model, the rate of uninhibited autolysis at zero DAPA concentration, 22 h-1, and the dissociation constant of the DAPA/meizothrombin complex, 5.9 x 10(-8) M, were obtained. The uninhibited autolysis rate so obtained was consistent with an estimate (19 h-1) obtained from gel filtration chromatography experiments. A surprising finding was that the data could not be described adequately without inclusion of a DAPA-insensitive, or background, rate of autolysis. We conclude that the active site of human meizothrombin can never be completely blocked and autolysis can never be completely prevented even in the presence of saturating concentrations of the active site inhibitor DAPA.

Activation/Inactivation of Human Factor V by Plasmin

The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.

Effect of thrombin inhibitors on thrombin-induced platelet release and aggregation

Thrombin-induced platelet activation was interrupted with hirudin or Dansylarginine N-(3-ethyl-1-5-pentanediyl)amide (DAPA) to study the time requirement for receptor occupancy by thrombin in promoting platelet responses at low (0.25 U/ml), intermediate (0.5 U/ml) and high (1 U/ml) thrombin concentrations. Each of these thrombin inhibitors suppressed adenosine triphosphate (ATP) release and aggregation by thrombin when added either before or simultaneously with thrombin or within seconds of the initiation of these responses by thrombin. If the inhibitors were added later, yet before aggregation or release was complete, no effect was present. The period of time for which active thrombin was required in order to promote these reactions had the following characteristics: (i) it is thrombin concentration dependent for a given response; (ii) it is longer for aggregation than for ATP secretion at each thrombin concentration; (iii) it is increased in platelets modified by chymotrypsin or platelets partially inhibited by antimycin A and 2-deoxy-D-glucose, which have prolonged aggregation and ATP release responses. In direct comparison studies, the inhibitory effects of hirudin and DAPA were identical on aggregation and ATP release. Thrombin binding, under similar experimental conditions identical to those, used to measure platelet activation, was prevented by hirudin, but not by DAPA. Therefore, the effect of DAPA on thrombin must be at the proteolytic site region and not at the hirudin-inhibitable platelet binding region. It is concluded from these studies that the tight coupling requirements for thrombin to induce platelet dense granule release and aggregation are directly dependent upon both the thrombin concentration and the rate of the individual platelet responses. Catalytic site integrity is required for the duration of this period of receptor occupancy.

Mechanism of potentiation of antithrombin III [at-III]inhibition by sulfated xylans

Anticoagulant properties of three sulfated compounds prepared from xylans isolated from corn cobs, larchwood and oatspelts were compared with heparin and sodium pentosan polysulfate (SP-54) by studying their effects on activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using pooled normal human plasma. These compounds were more effective than SP-54 in delaying coagulation by all the three procedures while oatspelts xylan sulfate was as effective as heparin in inhibiting APTT and PT and more effective than heparin in inhibiting TT on a molar basis. The sulfated xylans were more effective than heparin or SP-54 in potentiating the AT-III inhibition of amidolysis of H-D-Phe-Pip-Arg-pNa (S-2238) by thrombin (IIa) or amidolysis of Bz-Ile-Glu-Gly-Arg-pNa (S-2222) by Xa. Study of the high affinity binding of the xylan sulfates to AT-III-Sepharose column showed that the amount of the xylan sulfate recovered in the eluates from this peak was greatly increased with an increase in molecular weight (MW). A buffered mixture of IIa, AT-III and dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) was used to study the inactivation of IIa by AT-III. Larchwood xylan sulfate (2–10 μg) was found to accelerate this inactivation which was neutralized by human platelet factor 4(PF 4). The results also suggested an interaction between larchwood xylan sulfate and IIa which may potentiate an interaction between AT-III and IIa.

Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism.

The kinetics of the activation of human prothrombin catalyzed by human prothrombinase was studied using the fluorescent alpha-thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Prothrombinase proteolytically activates prothrombin to alpha-thrombin by cleavages at Arg273-Thr274 (bond A) and Arg322-Ile323 (bond B). The differential fluorescence properties of DAPA complexed with the intermediates and products of human prothrombin activation were exploited to study the kinetics of the individual bond cleavages in the zymogen. When the catalyst was composed of prothrombinase (human factor Xa, human factor Va, synthetic phospholipid vesicles, and calcium ion), initial velocity studies of alpha-thrombin formation indicated that the kinetic constants for the cleavage of bonds A or B were similar to the constants that were obtained for the overall reaction (bonds A + B). The progress of the reaction was also monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results indicated that the activation of human prothrombin catalyzed by prothrombinase proceeded exclusively via the formation of meizothrombin (bond B-cleaved) as an intermediate. Kinetic studies of the cofactor dependence of the rates of cleavage of the individual bonds indicated that, in the absence of the cofactor, cleavage at bond B would constitute the rate-limiting step in prothrombin activation. Progress curves for prothrombin activation catalyzed by prothrombinase and monitored using the fluorophore DAPA were typified by the appearance of a transient maximum, indicating the formation of meizothrombin as an intermediate. When factor Xa alone was the catalyst, progress curves were characterized by an initial burst phase, suggesting the rapid production of prethrombin 2 (bond A-cleaved) followed by its slow conversion to alpha-thrombin. Gel electrophoresis followed by autoradiography was used to confirm these results. Collectively, the results indicate that the activation of human prothrombin via the formation of meizothrombin as an intermediate is a consequence of the association of the cofactor, human factor Va, with the enzyme, human factor Xa, on the phospholipid surface.

The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction.

The activation of bovine prothrombin by prothrombinase (Factor Xa, Factor Va, synthetic phospholipid vesicles, and calcium ion) was studied in the presence of the fluorescent, reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA). Recordings of fluorescence intensity during prothrombin activation exhibited maxima that decreased to stable limiting values. These data suggested the transient appearance of the meizothrombin-DAPA complex, which exhibits fluorescence with 1.5-fold greater intensity than the thrombin-DAPA complex. At substrate concentrations well below Km, progress curves could be fitted by equations describing an ordered, sequential conversion of prothrombin to thrombin through the intermediate meizothrombin via two pseudo-first order steps. The pseudo-first order rate constants for both steps varied linearly with enzyme concentration, indicating that both steps are catalyzed by prothrombinase. The progress of the reaction was also monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry analyses of aliquots removed at intervals spanning the reaction. These analyses confirmed both the existence of meizothrombin and its time course as predicted from the equations used to analyze fluorescence intensity profiles. Meizothrombin levels peaked at about 0.3 mol/mol initial prothrombin under the conditions typically studied. In addition, prethrombin 2, which is the intermediate expected from cleavages occurring in the order opposite that required to form meizothrombin, was not observed under any of the conditions examined. These data indicate that prothrombin activation catalyzed by the fully assembled prothrombinase complex proceeds via an ordered, sequential reaction with meizothrombin as the sole intermediate.

Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide : A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase ( IC 50 = 2 × 10 −7 M) but not of acetylcholinesterase ( IC 50 = 4 × 10 −4 M) . For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K d of 4.5 × 10 −7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.

Cleavage and activation of human prothrombin by [formula omitted][formula omitted] venom

The cleavage of human prothrombin by partially purified Echis carinatus venom (ECV) was investigated in the present report. Incubation of prothrombin with ECV resulted in the rapid cleavage of prothrombin to alpha-thrombin, with the release of fragment-1 and fragment-2. When dansyl arginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), a very effective inhibitor of thrombin, was included in the ECV-prothrombin solution, meizothrombin was rapidly formed. Only small amounts of meizothrombin-1 could be detected. Prolonged incubation (23 h) in the presence of DAPA, however, resulted in nearly quantitative conversion of meizothrombin to meizothrombin-1 and fragment-1. Kinetic studies strongly suggested that the conversion of meizothrombin to meizothrombin-1 was due to ECV and not meizothrombin autolysis. In addition, EDTA, which inhibits ECV, blocked the cleavage of meizothrombin. Amino terminal sequence analysis indicated that ECV cleaves human prothrombin at two sites; Gly158-Ser159 and Arg322-Ile323. The former site differs from the site of autolytic cleavage of meizothrombin which occurs at Arg155-Ser156. In contrast to reports in the literature, the results of the present study indicate that the release of fragment-1 does not precede activation of human prothrombin by ECV.

Stroma free human platelet lysates potentiate the in vivo thrombogenicity of factor Xa by the provision of coagulant-active phospholipid.

A stroma free platelet lysate supernatant (PLS) was prepared by repeatedly freezing and thawing a human platelet suspension separated from platelet rich plasma by gel filtration. The material was potently thrombogenic in a stasis model in rabbits, but only when combined with a purified preparation of factor Xa. The dose of factor Xa used was not thrombogenic when given alone. Initial evaluation suggested the presence of a factor V activator when PLS shortened the clotting time of normal but not a V deficient plasma. Subsequent evaluation, in a system employing purified components of the prothrombinase complex and a marker of thrombin generation, dansyl-arginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA), failed to confirm the presence of a factor V activator. Further evaluation in this system demonstrated that the procoagulant activity related to the content of coagulant-active phospholipid. Thin layer chromatography confirmed the presence of phospholipids known to have coagulant activity. These components were isolated by a lipid extraction procedure and the phospholipid replacing activity in the in vitro assay with DAPA was quantitatively retained. The extracted material was non-thrombogenic in vivo but augmented the thrombogenicity of a purified preparation of Xa.

Studies on the Inhibition of Ellagic Acid-Activated Hageman Factor (Factor XII) and Hageman Factor Fragments

Hageman factor (HF, factor XII) that has been exposed to Sephadex-ellagic acid gels is a single-chain species (HFea) with amidolytic properties for the synthetic substrate H-d-phenylalanyl-l-pipecolyl-l-arginine p-nitroanilide. Earlier we reported that amidolysis was suppressed by incubation of HFea with specific antiserum. The present study provides additional evidence that the amidolytic properties of preparations of HFea are ascribable to this substance through an examination of a number of protease inhibitors. HFea’s amidolytic properties were inhibited by α2-plasmin inhibitor, antithrombin III in the presence of heparin, and Cl esterase inhibitor (Cl-INH). Additionally, it was inhibited by popcorn inhibitor, leupeptin, hexadimethrine bromide, protamine sulfate, dansyl-arginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA), diisopropylphosphofluoridate (DFP), aprotinin, and at excessively high concentrations, soybean and lima bean trypsin inhibitors. The spectrum of action of agents that did or did not inhibit HFea supports the view that amidolysis by preparations of HFea is attributable to this enzyme. In general, the enzymatically active carboxy-terminal fragment of HF (HFf) was inhibited by the same agents that inhibited HFea, but aprotinin, protamine sulfate and hexadimethrine bromide were more effective against HFf than HFea, while the reverse was true of lima bean trypsin inhibitor.