[14] Thrombin

Lundblad, Roger L.; Kingdon, Henry S.; Mann, Kenneth G.

doi:10.1016/s0076-6879(76)45017-6

Cited by 251 publications

(123 citation statements)

References 83 publications

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“…Recombinant ADAMTS13 containing a V5-His tag at the C terminus was expressed in HEK293 cells and purified according to published procedures (18). Thrombin was prepared from prothrombin and purified as described (28). Protein purity was assessed by SDS/PAGE under reducing conditions, followed by staining with Coomassie blue.…”

Section: Methodsmentioning

confidence: 99%

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Cao¹,

Krishnaswamy

Camire

et al. 2008

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

101

129

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Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VIII (FVIII) accelerates, by a factor of Ϸ10, the rate of specific cleavage at the Tyr 1605 -Met 1606 bond in VWF. Multimer analysis reveals that FVIII preferentially accelerates the cleavage of high-molecular-weight multimers. This rate enhancement is not observed with VWF predenatured with 1.5 M guanidine. The ability of FVIII to enhance VWF cleavage by AD-AMTS13 is rapidly lost after pretreatment of FVIII with thrombin. A FVIII derivative lacking most of the B domain behaves equivalently to full-length FVIII. In contrast, a derivative lacking both the B domain and the acidic region a3 that contributes to the highaffinity interaction of FVIII with VWF exhibits a greatly reduced ability to enhance VWF cleavage. Our data suggest that FVIII plays a role in regulating proteolytic processing of VWF by ADAMTS13 under shear stress, which depends on the high-affinity interaction between FVIII and its carrier protein, VWF.thrombotic thrombocytopenic purpura ͉ von Willebrand factor cleaving metalloprotease N ormal hemostasis requires the proteolytic processing of newly synthesized and secreted von Willebrand factor (VWF) in plasma by the metalloproteinase ADAMTS13. This process reduces VWF multimer size by cleavage at the Tyr 1605 -Met 1606 bond in the A2 subunit (1). Defective proteolytic processing of VWF multimers due to hereditary and/or acquired deficiency of ADAMTS13 activity results in the accumulation of ''unusually large'' multimers of VWF in plasma and disseminated microthrombi (2). These are characteristic pathological features of thrombotic thrombocytopenic purpura.ADAMTS13 cleaves VWF in a relatively inefficient way, and its preference for cleavage of the larger multimers is considered to lie in their greater susceptibility to deformation under shear stress or under flow while tethered to platelets (3). Previous studies have used a variety of manipulations to enhance cleavage including the use of ''low concentrations'' of urea (4) or guanidine hydrochloride (5) and prolonged incubation of denatured VWF with ADAMTS13 at low ionic strength, alkaline pH, and high concentrations of various divalent cations such as Ba 2ϩ , Ca 2ϩ , and Zn 2ϩ (1,4,6). These strategies are reminiscent of the extreme conditions used for observable cleavage of the coagulation zymogens in the absence of membranes and the appropriate cofactors that are now known to be essential for their efficient activation (7). Only very small enhancing effects of heparin and glycoprotein Ib on cleavage of denatured VWF have been reported previously (8). Thus, efficient processing of VWF by ADAMTS13 under more physiological conditions may be further regulated by a cofactor or by accessory components that are y...

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Section: Methodsmentioning

confidence: 99%

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Cao¹,

Krishnaswamy

Camire

et al. 2008

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

101

129

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“…Thrombin, prethrombin 1, and fragment 2 were proteolytically prepared from prothrombin and purified as described (12,14,29,30). Thrombin was chemically inactivated by treatment with FPR-CH 2 Cl to yield IIa i .…”

Section: Methodsmentioning

confidence: 99%

Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

Boskovic

Krishnaswamy²

2000

Journal of Biological Chemistry

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The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg 323 -Ile 324 , followed by cleavage at Arg 274 -Thr 275 . We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIa⌬F1) as a substrate analog to assess the mechanism of substrate recognition in the second half-reaction of bovine prothrombin activation. Cleavage of mIIa⌬F1 exhibits pseudo-first order kinetics regardless of the substrate concentration relative to K m . This phenomenon arises from competitive product inhibition by thrombin, which binds to prothrombinase with exactly the same affinity as mIIa⌬F1. As thrombin is known to bind to an exosite on prothrombinase, initial interactions at an exosite likely play a role in the enzyme-substrate interaction. Occupation of the active site of prothrombinase by a reversible inhibitor does not exclude the binding of mIIa⌬F1 to the enzyme. Specific recognition of mIIa⌬F1 is achieved through an initial bimolecular reaction with an enzymic exosite, followed by an active site docking step in an intramolecular reaction prior to bond cleavage. By alternate substrate studies, we have resolved the contributions of the individual binding steps to substrate affinity and catalysis. This pathway for substrate binding is identical to that previously determined with a substrate analog for the first half-reaction of prothrombin activation. We show that differences in the observed kinetic constants for the two cleavage reactions arise entirely from differences in the inferred equilibrium constant for the intramolecular binding step that permits elements surrounding the scissile bond to dock at the active site of prothrombinase. Therefore, substrate specificity is achieved by binding interactions with an enzymic exosite that tethers the protein substrate to prothrombinase and directs cleavage at two spatially distinct scissile bonds.Prothrombinase is an archetypal enzyme complex of blood coagulation (2). The enzyme complex assembles through well characterized, reversible, protein-protein and protein-membrane interactions between the serine protease, factor Xa, the cofactor, factor Va, and membranes in the presence of calcium ions (2-4). The resulting complex catalyzes the conversion of prothrombin to thrombin at a greatly enhanced rate, compared with the reaction rate catalyzed by factor Xa alone (2).Prothrombin is activated by proteolytic cleavage at two sites, Arg 274 -Thr 275 and Arg 323 -Ile 324 , which yields the fragment 1.2 activation peptide and thrombin 1 (5, 6). The reaction catalyzed by prothrombinase proceeds almost exclusively via the initial cleavage at Arg 323 -Ile 324 , yielding meizothrombin as an intermediate, followed by cleavage at Arg 274 -Thr 275 to yield the final products of the reaction (7,8). Single turnover kinetic studies indicate that the overall process is likely the sum of two consecutive enzyme-catalyzed reactions (8). Consequently, steady state...

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“…Purified human APC and factor Xa and dansylated Glu-Gly-Arg-chloromethyl ketone (DEGR) were generous gifts from Dr. Paul Haley, Hematologic Technologies, Inc. (Essex Junction, VT). Synthetic phospholipid vesicles composed of 75% L-palmitoyl-2-oleoyl phosphatidylcholine and 25% Lpalmitoyl 2-oleoyl phosphatidylserine (PCPS) and ␣-thrombin were prepared as described previously (38,39). Human factor V was purified and converted to the active form of the cofactor (Va) as described by Katzmann et al (40).…”

Section: Methodsmentioning

confidence: 99%

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

Long

Lu²,

Xie³

et al. 1998

Journal of Biological Chemistry

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Human factor Xa specifically cleaves the anticoagulant protein S within the thrombin-sensitive domain. Amino-terminal amino acid sequencing of the heavy chain cleavage product indicates cleavage of protein S by factor Xa at Arg 60 , a site that is distinct from those utilized by ␣-thrombin. Cleavage by factor Xa is unaffected by the presence of hirudin and is completely blocked by tick-anticoagulant-peptide and D-Glu-GlyArg-chloromethyl ketone, the latter two being specific inhibitors of factor Xa. The cleavage requires the presence of phospholipid and Ca 2؉ , and is markedly inhibited by the presence of factor Va. Factor Xa-cleaved protein S no longer possesses its activated protein C-dependent or -independent anticoagulant activity, as measured in a factor VIII-based activated partial thromboplastin time clot assay. The apparent binding constant for protein S binding to phospholipid (K d Ӎ 4 nM ؎ 1.0) is unaffected by factor Xa or thrombin cleavage, suggesting that the loss of anticoagulant activity resulting from cleavage is not primarily due to the loss of membrane binding ability. Cleavage and inactivation of protein S by factor Xa may be an additional way in which factor Xa exerts its procoagulant effect, after the initial stages of clot formation.Protein S is a vitamin K-dependent, 635-amino acid, nonenzymatic glycoprotein (M r Х 78,000; Ref. 1) that acts as an anticoagulant in blood (2-4). The domain organization of protein S is similar to other plasma vitamin K-dependent proteins in that it contains an amino-terminal ␥-carboxyglutamate-rich domain and several (four) epidermal growth factor-like domains (5, 6). Unique to protein S, however, is a 29-amino acid thrombin-sensitive domain, located between the ␥-carboxyglutamate and first epidermal growth factor-like domains. The thrombin-sensitive domain corresponds to exon IV of the protein S gene (7). Protein S also contains a sex steroid binding protein-like domain (8) at the carboxyl-terminal end of the protein, in place of the serine protease domain found in the vitamin K-dependent plasma proteases. The sex steroid binding protein-like domain has been identified as being composed of two tandem repeat units homologous to the Cys-poor laminin A globular (G) domain found in a number of extracellular ligand binding basement membrane proteins involved in cellular growth and differentiation (9). Schneider and co-workers have described a cultured cell growth arrest-specific protein (Gas6) from NIH 3T3 mouse cells (10) and human IMR90 fibroblasts (11), which, except for the absence of a thrombinsensitive domain, is homologous throughout to protein S.Thrombin cleavage converts protein S into a two-chain, disulfide-linked protein that no longer possesses anticoagulant activity (12). The thrombin cleavage sites were first established for bovine protein S (at Arg 52 and Arg 70 ) by Dahlback et al. (13) and recently at the corresponding residues (Arg 49 and Arg 70 ) for human protein S (1).The defined mechanism(s) by which protein S exhibits its anticoagulant acti...

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[14] Thrombin

Cited by 251 publications

References 83 publications

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Factor VIII accelerates proteolytic cleavage of von Willebrand factor by ADAMTS13

Exosite Binding Tethers the Macromolecular Substrate to the Prothrombinase Complex and Directs Cleavage at Two Spatially Distinct Sites

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

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