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Biomedical subjects

M E Nesheim

Publications and source records attributed to M E Nesheim.

At least 19 recordsLinked to original sources
PubMed1995

Factor V is complexed with multimerin in resting platelet lysates and colocalizes with multimerin in platelet alpha-granules.

Factor V stored in platelets is an important source of factor Va for the prothrombinase complex. Investigations of potential platelet factor Va-binding proteins, using factor Va light chain affinity chromatography, identified a disulfide-linked multimeric protein with a reduced mobility of 155 kDa in the column eluate. Immunodepletion and immunoblotting indicated that this protein was multimerin. Multimerin specifically bound factors V and Va and the isolated factor Va light chain, but not the heavy chain of factor Va. Factor V stored in platelets, but not plasma factor V, was found to be complexed with multimerin. Multimerin immunodepletion of resting platelet lysates was associated with the removal of factor V and the loss of factor V coagulant activity. Immunoelectron microscopic studies colocalized factor V with multimerin in the alpha-granules of resting platelets. With thrombin-induced platelet activation, we observed dissociation of factor Va-multimerin complexes, multimerin-independent membrane binding of factor Va, and prothrombinase activity that was not inhibitable by multimerin antibodies. This study indicates that platelet factor V is stored as a complex with multimerin and suggests a possible role for multimerin as a carrier protein for factor V stored in platelets.

Animals↗
PubMed1995

Prothrombinase components can accelerate tissue plasminogen activator-catalyzed plasminogen activation.

The enzymatic and cofactor subunits of human prothrombinase, factor Xa (FXa) and factor Va (FVa), respectively, were evaluated as modulators of Glu- and Lys-plasminogen (Pg) activation by tissue plasminogen activator (tPA). The data revealed that both FXa and FVa could accelerate tPA activity by as much as 60-fold for Lys-Pg and > 150-fold for Glu-Pg. This function of FVa depended on pretreatment with plasmin (Pn), whereas the FXa fibrinolytic cofactor activity was endogenous. In the native state, FVa was observed to inhibit the acceleration of Pn generation by FXa. These effects were dependent on Ca2+ and procoagulant phospholipid. Interactions between plasminogen and prothrombinase components were quantified. The apparent Kd for binding to FXa was 35 nM. Strikingly, the affinity between FVa and Pg was increased by approximately 2 orders of magnitude when the FVa was Pn-pretreated (Kd = 0.1 microM). These data cumulatively suggest a mechanism by which Pn production is coordinated with coagulation and localized to sites where procoagulant phospholipid is exposed on a cell surface.

Amino Acid Sequence↗
PubMed1995

Antifibrinolytic effect of recombinant apolipoprotein(a) in vitro is primarily due to attenuation of tPA-mediated Glu-plasminogen activation.

The effect of a 17-kringle form of recombinant apo(a) [r-apo(a)] on in vitro fibrin clot lysis was studied. In these assays, fibrin clots were formed in the wells of microtiter plates, and lysis of the clots was monitored by measurement of the turbidity at 405 nm. The results indicate that r-apo(a) produces a dose-dependent antifibrinolytic effect in clots formed using either purified components or barium-adsorbed plasma. This effect was found to be independent of clot structure, since lysis of clots formed using both high and low concentrations of thrombin was prolonged by r-apo(a) to the same extent. The two components of the antifibrinolytic effect of r-apo(a) were determined to be (i) attenuation of tPA-mediated plasminogen activation (the major component) and (ii) inhibition of plasmin degradation of fibrin, although r-apo(a) did not directly attenuate plasmin activity, as measured by S-2251 hydrolysis. r-Apo(a) interfered most substantially with tPA-mediated activation of Glu-plasminogen and less substantially with tPA-mediated Lys-plasminogen activation and urokinase-mediated activation of plasminogen. In summary, we have demonstrated that apo(a) is able to attenuate fibrin clot lysis in vitro, primarily as a consequence of the interference by apo(a) with tPA-mediated Glu-plasminogen activation. These studies illuminate possible mechanisms by which Lp(a) may contribute to the development of vascular disease in vivo.

Amino Acid Sequence↗
PubMed1993

Activation of protein C and its distribution between its inhibitors, protein C inhibitor, alpha 1-antitrypsin and alpha 2-macroglobulin, in patients with disseminated intravascular coagulation.

Activation and inactivation of protein C during the clinical course of disseminated intravascular coagulation (DIC) was studied in three patients by qualitative (Western blotting) and quantitative (ELISA) analysis and the intensity of procoagulant activity monitored by the measurement of thrombin and factor Xa antithrombin III complexes. In one patient, inhibitor complexes of APC with protein C inhibitor (PCI) and alpha 1-antitrypsin (alpha 1-AT) were observed and the latter predominated at presentation. Both disappeared during the development of remission but the loss of alpha 1-AT complexes preceded PCI complexes which on Western blotting appeared to increase in intensity prior to disappearance. The two other patients bled to death from uncontrollable haemorrhage. In both cases, APC/inhibitor complexes with alpha 2-macroglobulin (alpha 2-M) in addition to PCI and alpha 1-AT were detected and persisted until death. Although PCI appeared to be the primary inhibitor in all three cases, alpha 1-antitrypsin and particularly alpha 2-macroglobulin appeared to assume greater roles in the two fatal cases. These data are similar to previous findings in an experimental animal model of DIC that suggested that alpha 2-macroglobulin and alpha 1-antitrypsin become more important inhibitors of APC as the primary inhibitor PCI is consumed in the face of a sustained procoagulant challenge.

Adult↗
PubMed1992

Lys-plasminogen is a significant intermediate in the activation of Glu-plasminogen during fibrinolysis in vitro.

Plasminogen, the zymogen form of the fibrinolytic enzyme plasmin, is known to undergo plasmin-mediated modification in vitro. The modified form, Lys-plasminogen, is superior to the native Glu-plasminogen in fibrin binding and as a substrate for activation by tissue-type plasminogen activator (t-PA). The present study was undertaken to determine the existence and significance of the Glu- to Lys-plasminogen conversion during t-PA-mediated lysis of plasma clots in vitro. When human plasma was supplemented with exogenous Lys-plasminogen and clotted, a dose-dependent shortening of lysis time was observed. Formation of Lys-plasminogen in situ during fibrinolysis was determined using 131I-Glu-plasminogen-supplemented plasma. By the time of lysis, Lys-plasminogen had accumulated to about 20% of the initial concentration of Glu-plasminogen. Quantitation of activation of both Glu- and Lys-plasminogen as well as the conversion of Glu- to Lys-plasminogen in plasma supplemented with both 131I-Glu-plasminogen and 125I-Lys-plasminogen was accomplished by determining the flux of the isotopically labeled species along three pathways: Glu-plasminogen-->Glu-plasmin, Glu-plasminogen-->Lys-plasminogen, and Lys-plasminogen-->Lys-plasmin. After a brief lag, the Glu-plasminogen activation rate was constant until lysis was achieved, at which point activation ceased. The Lys-plasminogen activation rate also was essentially constant until lysis but was not characterized by a lag phase. The rate of conversion of Glu- to Lys-plasminogen was nonlinear and correlated directly with the rate of fibrinolysis. By the time lysis had occurred, Glu-plasminogen consumption had been distributed equally between direct activation to plasmin and conversion to Lys-plasminogen, and 45% of the plasmin which had been formed was derived from Lys-plasminogen. These results demonstrate both the formation and the subsequent activation of Lys-plasminogen during fibrinolysis. As a result of improved fibrin binding and activation of Lys-plasminogen compared to Glu-plasminogen, the formation of Lys-plasminogen within a clot constitutes a positive feedback mechanism that can further stimulate the activation of plasminogen by t-PA as fibrinolysis progresses.

Fibrinolysis↗
PubMed1992

Anticoagulant and fibrinolytic activities are promoted, not retarded, in vivo after thrombin generation in the presence of a monoclonal antibody that inhibits activation of protein C.

This study examines the assumption that both the anticoagulant and fibrinolytic activity that follow the generation of thrombin induced by infusion of factor Xa/PCPS are due to generation of activated protein C. Untreated controls or animals given unrelated antibody were compared with animals pretreated with a specific monoclonal antibody to protein C (HPC4). Compared with untreated controls excess HPC4 substantially reduced the level of protein C activation as observed by protein C immunoblotting and enzyme-linked immunosorbent assay for antitrypsin/activated protein C complexes. Despite this, the anticoagulant activity as reflected by the decline of factors Va and VIIIa levels (as observed by coagulation assays and by factor V immunoblotting) was significantly greater than controls. The fibrinolytic activity (as observed by assays of tissue plasminogen activator, D-Dimer, alpha 2-antiplasmin) also was significantly greater than controls. We conclude that neutralization of the protein C anticoagulant system while resulting in a significantly more intense coagulant response to Xa/PCPS does not preclude inactivation of factors Va and VIIIa and the full expression of the fibrinolytic response. We conclude further that after thrombin generation in vivo, protein C activation is not a prerequisite for the promotion of the fibrinolytic response previously observed, and that the inactivation of factors Va/VIIIa may be mediated by enzymes other than activated protein C. The reduction in alpha 2-antiplasmin levels in association with increased tissue plasminogen activator activity suggests that plasmin is a likely candidate.

Animals↗
PubMed1992

Platelet factor Xa receptor.

The assembly and function of the prothrombinase complex on the bovine and human platelet membrane is mediated through binding interactions in which factor Va bound to the platelet surface forms at least part of the "receptor" for factor Xa in a 1:1 stoichiometric complex. A model depicting these binding interactions is shown in Fig. 12. Data from our laboratory indicate that the prothrombinase catalyst assembles in an analogous manner on the surface of monocytes, lymphocytes, neutrophils, and well-defined phospholipid vesicles employed in model systems. The 74,000-Da subunit of factor Va, component E, which mediates the binding of factor Va to either bovine platelets, human monocytes, or phospholipid vesicles, is shown binding to the cell membrane through its putative "receptor." The 94,000-Da subunit of factor Va, component D, is associated with the membrane surface through its metal ion-dependent interaction with component E. Factor Va forms at least part of the receptor that mediates the binding of factor Xa to an appropriate membrane surface, because component E has been shown to contribute significantly to the interaction of factor Xa with either the platelet, monocyte, or vesicle membrane surface. Our data do not preclude the possibility that component D contributes to the binding of factor Xa and the function of the prothrombinase complex. Component D appears to be important for several reasons. Cleavage of component D by activated protein C results in the complete loss of factor Va cofactor activity. An interaction between factor Xa and component D is implied from the observation that factor Xa protects factor Va from activated protein C inactivation. Furthermore, the binding of factor Xa to platelet-bound factor Va results in the time-dependent cleavage of components D and D'. Because component D is not required absolutely for prothrombinase complex assembly, we would speculate that it may be important in mediating prothrombin binding (depicted as a three-domain molecule) and increasing the catalytic efficiency of the enzymatic complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Alpha 2-macroglobulin binds and inhibits activated protein C.

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine-Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed1991

Kinetic characterization of tissue-type plasminogen activator (t-PA) and t-PA deletion mutants.

The binding of t-PA to fibrin is mediated both by its "finger" (F) and its "kringle 2" (K2) domain. In addition, these domains are involved in the stimulation of t-PA activity by fibrin. We analyzed the kinetic characteristics of Glu-plasminogen activation by t-PA and a set of t-PA deletion mutants in the absence and the presence of desA-fibrin. In the absence of desA-fibrin, the activity of t-PA (variants) is determined by the presence of the protease domain, irrespective of the composition of the amino-terminal heavy chain. In the presence of the cofactor desA-fibrin, the activity of t-PA (variants) is dependent on the domain composition of the heavy chain. The activity of t-PA is stimulated 2,400 fold by desA-fibrin, whereas the activity of the mutant lacking the K1 domain (del. K1) increases 936 fold in the presence of this cofactor. Mutants lacking either the K2 domain (del. K2) or the F domain (del. F) exhibit an enhanced activity upon desA-fibrin addition of 200 and 210 fold, respectively. DesA-fibrin has no stimulatory effect on the activity of the mutant containing only the serine-protease domain (del.FE K1 K2) nor on the activity of the variant containing only the K1 domain and the serine-protease domain (del. FE K2). Furthermore, we determined the relative fibrin affinity of each t-PA variant, which is similarly dictated by the composition of the heavy chain.

Amino Acid Sequence↗
PubMed1990

Surface-dependent reactions of the vitamin K-dependent enzyme complexes.

During the past 20 years contributions from many laboratories have led to the development of isolation procedures, delineation of primary structures, and more recently, to the expression of recombinant proteins associated with the coagulation cascade. In general, studies of coagulation proteins under defined conditions have demonstrated the prescience of Davie and Ratnoff and MacFarlane in their proposals of the coagulation cascade. The more recent discovery of thrombomodulin by Esmon et al has led to the identification and characterization of components of the vitamin K-dependent anticoagulant pathway. In this review we have attempted to analyze and compare the functional properties of each of the vitamin K-dependent enzyme complexes associated with the procoagulant and anticoagulant phases of blood clotting. Although dissimilarities exist, the vitamin K-dependent complexes have analogous requirements and appear to function with a common general mode of organization. Membrane-bound cofactors serve as anchoring sites for the appropriate membrane-binding enzymes. This process localizes the complex on the membrane surface and increases the catalytic efficiency for substrate utilization. Complex formation provides extraordinary improvements in the catalytic efficiency for the complexes as compared with their soluble enzyme components. Membrane-bound complexes provide a mechanism that can be regulated at a site by membrane presentation, zymogen activation, and cofactor activation or presentation. The kinetic constants obtained for the various coagulation reactions determined in vitro provide some insights into how these pathways may function in vivo. The catalytic efficiency (kcat/Km) for factor X activation by factor VIIIa/factor IXa is far in excess of the catalytic efficiency of activation of factor X by tissue factor/factor VIIa (Table 3). This may provide a rational interpretation for the observation that patients with hemophilia A and B bleed even though they appear to have an alternative pathway to factor X activation. In addition, tissue factor is not ordinarily presented by the vascular tissue that has direct access to blood. However, it appears that extravascular constitutive tissue factor is available once the blood vessel becomes disrupted. The efforts to identify the initiating reactions of the blood coagulation process have not been unambiguously successful. We conclude that factor VII is most likely a zymogen, just as are the other proenzymes of the blood clotting process. In addition, it is difficult to rationalize the importance of the intrinsic pathway of coagulation involving factor XII, prekallikrein, and high molecular weight kininogen since the congenital absence of any one of these factors does not result in abnormal bleeding.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Coagulation↗
PubMed1990

The fibrinolytic potential of the normal primate following the generation of thrombin in vivo.

Parameters of the fibrinolytic system were studied in a primate model where the generation of thrombin was promoted in vivo. The procoagulant stimulus used was a combination of human factor Xa in combination with phosphatidylcholine/phosphatidylserine lipid vesicles (PCPS) as the source of coagulant active phospholipid. The dosage of each component was formulated to provide a gradation of thrombin generating potential assessed prior to in vivo study in an in vitro clotting assay. These ranged from 25.25-36.60 pMole/kg (factor Xa) and 18.85-56.30 nMole/kg (PCPS). In each case, the ratio of the dose of factor Xa/PCPS was maintained at 0.65 (pMole factor Xa/nMole PCPS). Individual dosage combinations producing recalcification clotting times in vitro of 15, 20, 25 and 30 s were used in detailed in vivo studies. Previous studies in dogs had confirmed the thrombin generating potential of factor Xa/PCPS infusions and demonstrated an associated activation of protein C and increased fibrinolytic activity. This has now been extensively characterized in the chimpanzee as follows: 10 min after the infusion of the highest dose (36.6 pMole factor Xa/56.3 nMole PCPS kg bodyweight), the level of circulating t-PA had risen to 900 ng/ml (antigen), 885 IU/ml (functional). Dosage was observed with the lowest dose of 12.25 pMole factor Xa and 18.85 nMole PCPS being associated with relatively minor increases in circulating t-PA activity. There were no changes in u-PA at any dosage during the full time course of the experimental period (90 min). Plasminogen activation was also apparent with alpha-2 antiplasmin levels falling to 30-40% of pre-infusion levels at the highest dosages.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid.

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.

Amino Acid Sequence↗
PubMed1990

A qualitative and quantitative analysis of the activation and inactivation of protein C in vivo in a primate model.

A model of Protein C (PC) activation in vivo was used to investigate the complexing of activated PC (APC) with its plasma inhibitors, PC inhibitor (PCI) and alpha 1-antitrypsin (alpha 1AT). Chimpanzees were infused with a bolus of activated factor X (F.Xa) together with vesicles of phosphatidylcholine and phosphatidylserine (PCPS). Pre- and post-infusion plasma samples were analyzed using enzyme linked immunosorbent based assays (ELISA) for PC and APC complexes, and immunoblotting of PC from nondenaturing polyacrylamide gel electrophoresis. Within 2 minutes of infusion, a 60% decrease in nonactivated PC zymogen (PCz) levels was observed. This coincided with a precipitous drop in plasma activities of cofactors VIIIa and Va. In contrast, total PC antigen (PCt) levels decreased by only 1%, indicating APC generation. Complexes of APC with both PCI and alpha 1AT were observed on immunoblots, and further identified and quantified using a sandwich ELISA employing antibodies to both PC and these inhibitors. The distribution of APC between these two inhibitors varied with the dose of F.Xa/PCPS infused. At a dose of F.Xa/PCPS of 24.05 pmol and 37.70 nmol/kg, respectively, an initial spike of APC generation, associated with decreases in the levels of factors VIIIa and Va, was noted but dissipated over the next 30 minutes. During this period, APC/inhibitor complexes appeared with the levels of APC-PCI and APC-alpha 1AT reaching 8.5 nmol/L and 2.2 nmol/L by 30 minutes, respectively. In contrast, at a higher dose of F.Xa/PCPS of 36.60 pmol and 56.30 nmol/Kg respectively, complexes of APC-alpha 1AT appeared rapidly and reached a level of 6 nmol/L by 30 minutes postinfusion, whereas APC-PCI complexes were only present at a concentration of 3.4 nmol/L by this time. Additional experiments with lower doses of F.Xa/PCPS suggest that PCI is the preferred inhibitor of APC, but as the availability of this inhibitor becomes limiting, alpha 1AT plays an increasingly crucial role as a secondary inhibitor of endogenously generated APC. Moreover, evidence is presented suggesting the existence of additional inhibitor(s) of APC that may have a role similar to alpha 1AT.

Animals↗
PubMed1988

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets.

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.

Blood Platelets↗
PubMed1988

Evidence that the thrombin-catalyzed feedback cleavage of fragment 1.2 at Arg154-Ser155 promotes the release of thrombin from the catalytic surface during the activation of bovine prothrombin.

During the course of prothrombin activation, as catalyzed by Factor Xa, Factor Va, Ca2+, and negatively-charged phospholipid vesicles, the three proteins distribute between the fluid phase and the vesicle surface. On the vesicle, efficient Factor Xa-catalyzed proteolysis yields thrombin plus Fragment 1.2. Further thrombin-catalyzed feedback cleavage of the latter then yields Fragment 1 plus Fragment 2. Prior to this cleavage Fragment 1.2 might retain thrombin at the site of catalysis since it binds both phospholipid and thrombin through its respective Fragment 1 and Fragment 2 domains. In order to study the role of the feedback cleavage, light scattering at right angles was used to deduce the nature of the components associated with the vesicle during prothrombin activation by continuous monitoring of the relative molecular weight of the vesicle-protein complex. When prothrombin (1.4 microM) was added to homogeneously sized phospholipid vesicles of phosphatidylcholine-phosphatidylserine (3:1) at a total phospholipid concentration of 20 microM, the scattering intensity doubled. Upon subsequent addition of Factor Xa and Factor Va (5.0 nM each) the scattering intensity smoothly decreased to a value about 1.25-fold greater than that of the vesicles alone. Analysis of the composition of the reaction mixture at intervals during the course of the reaction by gel electrophoresis and laser densitometry, provided a good correlation between the mass of the vesicle-protein complex measured by light scattering and its mass inferred by composition. In addition, the decrease in mass of the vesicle-protein complex measured by light scattering correlated temporally with cleavage of Fragment 1.2. When the reaction was initiated in the presence of the reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide no cleavage of Fragment 1.2 occurred, as indicated by gel electrophoresis, and no change in the mass of the vesicle-protein complex occurred as indicated by light scattering. The absence of change in scattering intensity in the presence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide suggests a 1:1 replacement of prothrombin at the catalytic surface by components of equivalent mass (Fragment 1.2 plus thrombin), whereas the decrease in scattering in the absence of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide suggests replacement of prothrombin by Fragment 1 only. Together these results indicate that the thrombin-catalyzed cleavage of Fragment 1.2 promotes release of thrombin from the catalytic surface.

Algorithms↗
PubMed1988

A combination of factor Xa and phosphatidylcholine-phosphatidylserine vesicles bypasses factor VIII in vivo.

A combination of phosphatidylcholine-phosphatidylserine lipid vesicles (PCPS), as a source of coagulant active phospholipid, when infused with factor Xa bypasses factor VIII in vivo. To demonstrate this, a reproducible model of bleeding in haemophilic dogs was used. Control studies were performed in normal dogs. In initial studies, factor Xa/PCPS at a dose of 6.5 x 10(-12) and 4.0 x 10(-7) moles/kg respectively failed to correct the abnormal bleeding in the haemophilic animals and initiated a bleeding diathesis in the normal controls. Coagulation studies and immunoblotting demonstrated activation of protein C and an anticoagulant effect resulting from significant falls in the levels of factors V and VIII. Adjustment of the dose of factor Xa/PCPS to 2.6 x 10(-11) and 4.0 x 10(-8) moles/kg respectively produced an immediate haemostatic effect in both haemophilic and normal animals with bleeding stopping within 15-30 s. Despite this observation, protein C activation was again noted. It is concluded that the presence of coagulant active phospholipid and factor Xa in prothrombin complex concentrates may explain the observed factor VIII bypassing activity of these preparations and that the use of a controlled formulation of these two components may provide a more effective approach to the management of patients with factor VIII inhibitors.

Animals↗