Inhibition of human blood coagulation factor Xa by .alpha.2-macroglobulin

Meijers, Joost C. M.; Tijburg, P; Bouma, Bonno N.

doi:10.1021/bi00392a053

Cited by 37 publications

(25 citation statements)

References 29 publications

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“…Strikingly, the fibrinolytic activity is decreased during this period. In the present work, we demonstrate by PAGE analysis that PZP interacts with t-PA within 1 h of incubation at room temperature, whereas a 2 -M reacts over a longer period of up to 18 h. Formation of a -macroglobulint-PA complexes was evidenced by a number of observations, as follows: (a) PZP and <z 2 -M showed changes in their mobility rates in non-denaturing PAGE comparable to those observed with chymotrypsin (2,3,28); (b) both PZP and a 2 -M, formed complexes of molecular size >360 kDa in agreement with their covalent binding to t-PA, as reported for other proteinases (30)(31)(32); and (c) PZP underwent a specific cleavage of the bait region with rapid appearance of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, this proteolytic attack on a 2 -M as found to be slow, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa (2,28).…”

Section: Discussionsupporting

confidence: 68%

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Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction

Sánchez¹,

Chiabrando

Guglielmone

et al. 1998

Journal of Biochemistry

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Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on alpha2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of alpha-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of alpha2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.

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Section: Discussionsupporting

confidence: 68%

“…Early studies showed that a 2 -M plays a key role in the regulation of both coagulation and fibrinolysis (32,(41)(42)(43). In this context, Arbelaez et al (44) reported that PZP exerts inhibition on human tissue kallikrein, but not on other proteinases.…”

Section: Discussionmentioning

confidence: 99%

Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction

Sánchez¹,

Chiabrando

Guglielmone

et al. 1998

Journal of Biochemistry

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“…Since a2-macroglobulin is capable of inhibiting a broad spectrum of proteolytic enzymes, it may be increased during hibernation to reduce proteolysis in a general way. Alternatively, a2-macroglobulin may play a specific role in the inhibition of blood clotting; a2-macroglobulin is known to be the main physiological inhibitor ofactivated factor X, a key enzyme in both the intrinsic and extrinsic clotting pathways (22). It may be important to reduce clotting during hibernation because of the low heart rate.…”

Section: Resultsmentioning

confidence: 99%

Central role for differential gene expression in mammalian hibernation.

Srere

Wang

Martin

1992

Proc. Natl. Acad. Sci. U.S.A.

144

105

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Mammalian hibernators experience dramatic reductions in body temperature, metabolic rate, respiratory rate, and heart rate during hibernation. These changes are precisely controlled and reversible with only internally driven mechanisms, suggesting specific biochemical regulation. We present a model that integrates our observations of differential liver gene expression during preparation for, and maintenance of, the hibernating state, with the known phylogenetic interspersion of hibernating species in several major mammalian lineages. This model predicts a major role for the differential expression of existing mammalian genes in the biochemical regulation of hibernation.Hibernation is an adaptive strategy that is used by species in several mammalian orders to conserve energy in cold or inhospitable environments. During hibernation, these mammals dramatically lower their metabolic, heart, and respiratory rates as well as their body temperature in a precisely controlled manner. This strategy helps some, particularly small, mammals to survive cold winter conditions in temperate climates when food and water are scarce yet the demand for metabolic heat generation is high. Hibernating species are frequently closely related to nonhibernating species and are found in five orders of eutherian mammals as well as in a few species of marsupials and monotremes (ref. 1 and Fig. 1). The adaptive significance of hibernation, coupled with the interspersed phylogenetic distribution of hibernating species among mammals, makes hibernation an ideal model system to test the hypothesis that the molecular basis of important adaptive events during evolution involves mechanisms that lead to the differential regulation of existing genes and not the invention of new genes (reviewed in ref.3).The evolutionary origin of hibernation is unknown; either of two hypotheses can explain the interspersed nature of the distribution of extant hibernating species throughout Mammalia. One postulates that the common ancestor of all mammals was a hibernator, but the ability to hibernate was lost independently along multiple lineages. The alternative proposes that the common ancestor of all mammals was not a hibernator, and the ability to hibernate has been gained independently along multiple lineages (Fig. 1). With either scenario, we predict that a small number of regulatory changes determine the phenotype of hibernation, rather than the de novo creation or loss of numerous hibernation-specific biochemical functions, encoded by a collection of hibernation-specific genes. Thus, hibernation must be the result of a reprogramming of existing mammalian biochemical capabilities through the differential expression of existing genes.There is little evidence in the literature for differential gene expression during hibernation, although there are a few reports that demonstrate increases or decreases (4) in the amount of specific proteins relative to the active state. Mitochondrial uncoupling protein (thermogenin) is an example of a protein that increases i...

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“…Purified human factor Xa was a generous gift of Dr J. C. M. Meijers from our laboratory [28]. This material contained no detectable prothrombin or thrombin as judged by gel electrophoresis.…”

Section: Proteinsmentioning

confidence: 99%

The effect of von Willebrand factor on activation of factor VIII by factor Xa

Koedam¹,

Hamer²,

Beeser‐Visser³

et al. 1990

European Journal of Biochemistry

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Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 Ujml) with 0.1 pg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 pg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 pg/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12,20/21,23,27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.Factor VIII is an important plasma protein which acts as a cofactor in the intrinsic pathway of coagulation. It does so by dramatically increasing the V,,, for the activation of factor X by the enzyme factor IXa in the presence of calcium and a phospholipid surface [I]. The critical role of factor VIII in hemostasis is illustrated by the severe bleeding tendency (known as hemophilia A) in its absence [2]. It is a key protein in the regulation of the coagulation cascade, since its cofactor activity can be enhanced through feed-back mechanisms by thrombin and factor Xa. Factor VIII activity is subsequently destroyed by the regulatory-enzyme-activated protein C. Both activation and inactivation of factor VIII occur through specific proteolytic cleavages within the molecule [3 -51.Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6, 71 and purified from plasma as a carboxy-terminal light chain of 80 kDa which is associated with an amino-terminal heavy chain ranging in molecular mass between 90 -210 kDa [4, 8 -111. In many studies, the kinetics of factor-VIII and factor-X activation [I, 12-181, as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3,4, 17, 191, have been described. These studies have ignored, however, that factor VIII circulates as a non-covalent complex with von Willebrand factor [20]. Formation of this complex is of great physiological importance for factor VIII, since it prolongs its half-life in the circulation [21,22]. Since von Willebrand factor is a very large glycoprotein which represents 99% of the mass of the factor-VIII -von-Willebrand-factor complex, it is conceivable that it modulates the activation and inactivation of factor VIII.In a previous paper ...

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Inhibition of human blood coagulation factor Xa by .alpha.2-macroglobulin

Cited by 37 publications

References 29 publications

Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction

Interaction of Human Tissue Plasminogen Activator (t-PA) with Pregnancy Zone Protein: A Comparative Study with t-PA- 2 -Macroglobulin Interaction

Central role for differential gene expression in mammalian hibernation.

The effect of von Willebrand factor on activation of factor VIII by factor Xa

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