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PubMed1999

Platelet responses to compound interactions with thrombin.

Catalytic and noncatalytic interactions of thrombin with platelets are investigated with use of thrombin variants with altered specificities and with ligands of thrombin receptors on platelets. Both alpha-thrombin and weakly coagulant meizothrombin-des-fragment-1 (mu-thrombin) hydrolyze proteolytically activated receptor 1 for thrombin (rPAR1(T), recombinant) with catalytic efficiencies of >10(7) M(-)(1) s(-)(1), whereas rPAR1(T) is not a substrate for weakly coagulant beta-thrombin. In contrast, both mu-thrombin and beta-thrombin are weak agonists of platelet dense body (ATP) secretion. Antibodies that block rPAR1(T) cleavage strongly inhibit the secretory reaction to alpha- and mu-thrombins but not to beta-thrombin or to thrombin receptor activating peptide (TRAP). However, catalytically inactive FPR-thrombin, which binds glycoprotein Ib but does not inhibit rPAR1(T) cleavage, inhibits responses to TRAP as well as those to alpha- and mu-thrombins, which indicates that binding of the inactive enzyme to platelets influences the function of PAR1(T). An antibody that inhibits binding of thrombin to platelet glycoprotein Ib inhibits secretory responses to thrombin but not to TRAP, so occupancy of glycoprotein Ib per se accounts for only part of the attenuation. All three thrombins stimulate a rise in cytosolic Ca(II), and the dose response to beta-thrombin is congruent with that for ATP secretion. However, the response of cytosolic Ca(II) is 10-100 times more sensitive to mu-thrombin and alpha-thrombin than ATP secretion is, and is inhibited by neither anti-PAR1(T) Ig nor FPR-thrombin. Thus, alpha-thrombin appears to have an activity not shared by either mu- or beta-thrombins. This activity is owed to more than coupling of independent signals from cleavage of two proteolytically activated receptors, as there is no synergism when mu-thrombin and beta-thrombin costimulate secretion. It is concluded either that alpha-thrombin has a third interaction site on platelets with which neither mu-thrombin nor beta-thrombin interacts or that dual receptors are coordinately cleaved. In either case, the strong secretory response to thrombin appears to be moderated, independently of cytosolic Ca(II), by occupancy of a noncatalytic interaction site such as glycoprotein Ib.

Adenosine Triphosphate↗
PubMed1997

Contribution of lysine 60f to S1' specificity of thrombin.

Lys60f has been proposed to limit the S1' substrate binding site specificity of thrombin to small polar P1' residues by occluding the S1' binding pocket, based on the X-ray crystal structure of thrombin. To test this proposal, we prepared a Lys-->Ala (K60fA) mutant of recombinant thrombin and determined whether this mutation enhanced the reactivity of thrombin with a variant inhibitor [antithrombin (AT)-Denver] and a substrate (protein C) containing poorly recognized P1' Leu residues. AT-Denver in the presence of heparin inhibited K60fA thrombin with a second-order association rate constant [k = 4.2 +/- 0.1) x 10(5) M-1 s-1] that was 3.2-fold faster than thrombin [k = (1.3 +/- 0.1) x 10(5) M-1 s-1]. Wildtype AT (P1' Ser) under the same conditions inhibited K60fA thrombin with a 2.5-fold slower rate constant [k = (1.1 +/- 0.1) x 10(7) M-1 s-1] than thrombin [k = (2.8 +/- 0.1) x 10(7) M-1 s-1]. These results indicate an overall 8.3-fold improvement in the recognition of the P1' Leu of AT-Denver by K60fA thrombin over that of wild-type thrombin; i.e., the K60fA mutation partly overcomes the defect in thrombin inhibition produced by the P1' mutation in AT-Denver. Resolution of the two-step reactions of AT and AT-Denver with wild-type and mutant thrombins revealed that the enhanced recognition of P1' Leu in AT-Denver by K60fA thrombin occurs primarily in the second reaction step in which a noncovalent AT-thrombin encounter complex is converted to a stable, covalent complex. Thrombin K60fA activated Gla-domainless protein C (GDPC) approximately 2- and approximately 4-fold faster than thrombin in the presence and absence of thrombomodulin (TM), respectively, consistent with an improved interaction of the Leu P1' residue with the mutant S1' pocket. In contrast, the mutant thrombin clotted fibrinogen (P1' Gly) approximately 3-fold slower than thrombin. Kinetic analysis revealed that the improvement in the catalytic rate of activation of GDPC by K60fA thrombin in the presence of TM was localized in the second reaction step, as reflected by an approximately 2-fold increase in kcat. Direct binding studies showed that the K60fA mutation minimally affected the affinity of thrombin for Na+, indicating that the changes in S1' site-specificity of K60fA thrombin did not result from altering the allosteric transition induced by Na+. We conclude that Lys60f limits the P1' substrate and inhibitor specificity of thrombin by influencing the size and polarity of the S1' site which thereby affects the stability of the transition state for cleavage of the scissile bond in the second reaction step.

Binding Sites↗
PubMed1993

Thrombin regulates tissue factor and thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms.

The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence thrombomodulin mRNA levels. In contrast, surface-bound thrombomodulin cofactor activity and thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged expression on the cell surface.

Amino Acid Chloromethyl Ketones↗
PubMed1993

The chondroitin sulfate moiety of thrombomodulin binds a second molecule of thrombin.

The role of the chondroitin sulfate moiety of thrombomodulin (TM) in the binding of thrombin to TM has been examined using fluorescent derivatives of thrombin. An anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg (FPR) linkage to form ANS-FPR-thrombin. When ANS-FPR-thrombin was titrated with TM lacking the chondroitin sulfate moiety (csf-TM), a monotonic and saturable increase in ANS emission intensity was observed that was consistent with the formation of a high affinity 1:1 thrombin-csf-TM complex. In contrast, titration of ANS-FPR-thrombin with intact TM containing the chondroitin sulfate resulted in a biphasic change in ANS-FPR-thrombin emission intensity that was consistent with each molecule of TM binding at least two molecules of ANS-FPR-thrombin with different affinities. This suggested that the second thrombin binds to TM via the chondroitin sulfate moiety. A direct interaction between thrombin and chondroitin sulfate was demonstrated by showing that chondroitin sulfate, cleaved and purified from TM, caused a saturable increase in ANS emission intensity upon addition to an ANS-FPR-thrombin sample. This spectral change was reversed by adding an excess of unmodified thrombin. The minimum Kd for the ANS-FPR-thrombin-chondroitin sulfate complex was approximately 20 nM, consistent with chondroitin sulfate being the lower affinity binding site on TM for thrombin. The titration of chondroitin sulfate into ANS-FPR-thrombin samples in the absence and presence of a TM fragment containing the fifth and sixth growth factor-like domains (GF5-6) showed that GF5-6 did not block chondroitin sulfate binding and that a GF5-6-thrombin-chondroitin sulfate ternary complex was formed. Thus, the chondroitin sulfate binds to thrombin somewhere other than anion-binding exosite I, and in doing so, alters the structure and/or environment of the active site more than 15A from the active site serine without detectably changing the conformation near Ser-195. Since excess TM and excess csf-TM increased the ANS emission intensity of ANS-FPR-thrombin to different extents (approximately 15 and approximately 80%, respectively), the chondroitin sulfate also influences the environment of the active site probe even when thrombin is bound to the higher affinity site on TM (GF5-6).

Antithrombin III↗