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PubMed1995

Effects of heparin and hirudin on thrombin generation and platelet aggregation after intrinsic activation of platelet rich plasma.

The effects of unfractionated heparin (UH) and recombinant hirudin (rH) on prothrombin activation, free thrombin generation, and platelet aggregation induced by endogenously generated thrombin after intrinsic activation of platelet rich plasma were compared. Free thrombin generation and platelet aggregation were assessed simultaneously by delaying fibrinogen polymerisation with GPRP. UH more effectively inhibited prothrombin activation and free thrombin generation than rH. Increasing concentrations of rH had hardly any effect on the peak amount of free thrombin, while in the presence of 400 nM UH only traces of free thrombin were detected. Comparison of TAT and THC (thrombin-hirudin complex) generated until the onset of platelet aggregation on a molar basis showed that much more thrombin was inactivated in the presence of rH than in plasma containing UH. The explosive generation of free thrombin in hirudinized plasmas was accompanied by a markedly steeper aggregation curve as compared to heparinized plasmas. The generation of thromboxane B2 was markedly delayed in the presence of UH but not influenced in the presence of rH. Our results suggest that UH is more effective than rH in inhibiting prothrombin activation after intrinsic activation of platelet rich plasma, while rH prevents clotting more by direct inactivation of already generated thrombin. The inability of even high concentrations of rH to prevent the explosive generation of free thrombin might contribute to the observed inefficiency of rH to inhibit platelet aggregation.

Blood Platelets↗
PubMed1979

[Participation of the hypophyseal-adrenal cortex system in thrombin clearance during immobilization stress].

The examination carried out with thrombin marked by 131J resulted in a considerable increase of the thrombin clearance rate in healty male rats during the stress (caused by an immobilization lasting 30 minutes) and in an increase of thrombin deposits in the liver. A further increase of thrombin clearance occurred by the combination of immobilization and administration of ACTH. Contrary to ACTH the thrombin clearance is not stimulated in healthy animals by hydrocortisone. Thrombin clearance and thrombin deposits in the liver are lowered in adrenalectomized rats. In these animals the administration of ACTH does not result in an increase of thrombin clearance. The rate of thrombin clearance is normalized in adrenalectomized animals after administering hydrocortisone without as well as under conditions of stress. In adrenalectomized animals having received hydrocortisone as well as in healthy animals the administration of ACTH will results in an increase of thrombin clearance. From these experiments the conclusion can be drawn that ACTH will increase the intensity of thrombin clearance in stress and that hydrocortisone plays a transmitting part here.

Adrenal Cortex↗
PubMed1997

Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex.

We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 micromol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti-GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.

Animals↗
PubMed1997

Inhibition of thrombin generation in plasma by inhibitors of factor Xa.

A series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations. The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

Animals↗
PubMed1998

Effects of thrombin on the growth, protein synthesis, attachment, clustering and alkaline phosphatase activity of cultured human periodontal ligament fibroblasts.

Previous studies have indicated that thrombin can activate pulp cells, including fibroblasts. Because pulp cells and periodontal ligament (PDL) fibroblasts can express thrombin receptor mRNA, the specific aim of this study was to determine whether thrombin can activate the growth, attachment, protein synthesis, alkaline phosphatase activities and cellular clustering of cultured human PDL fibroblasts. Thrombin can stimulate the growth of PDL fibroblasts in a dose dependent manner (as analyzed by MTT assay). At concentrations of 5 and 10 U/ml, thrombin increased the cell numbers to 141% and 153% greater than that of the control after 5 days of incubation, respectively. Thrombin (5-20 U/ml) also stimulated the protein synthesis rate (assayed by [3H]proline incorporation) to 1.88-2.13 fold that of the control. However, pretreatment of PDL fibroblasts with thrombin (1-20 U/ml) could not promote the attachment of PDL fibroblasts to type I collagen and fibronectin. Moreover, thrombin could induce clustering of PDL fibroblasts within a concentration range of 5-20 U/ml. However, thrombin (1-20 U/ml) exerted neither stimulatory nor inhibitory effect on cellular alkaline phosphatase activities. In conclusion, it appears that the presence of thrombin seems to have effects on PDL fibroblasts in terms of cell growth, protein synthesis and cell clustering. This suggests that thrombin might be important in the early healing process of periodontium following periodontal surgery.

Alkaline Phosphatase↗
PubMed2002

Anti-thrombin is expressed in the benign prostatic epithelium and in prostate cancer and is capable of forming complexes with prostate-specific antigen and human glandular kallikrein 2.

Anti-thrombin, a member of the serpin family and an inhibitor of thrombin and blood coagulation factor Xa, was recently shown to inhibit angiogenesis and tumor growth. In the present study, we examined the expression of anti-thrombin in benign and malignant prostate gland. Using immunohistochemistry, anti-thrombin was found in prostate epithelium and stroma cells. Tissue microarrays of tumors (n = 112) and three different prostate cancer cell lines (PC-3, LNCaP, and DU-145) were all positive for anti-thrombin. Abundant expression in a population of prostatic tumor cells was further evidenced by in situ hybridization experiments. The immunostaining for anti-thrombin was confined to the cytoplasm, was most intense in Gleason grade 3 tumors, and in part overlapped with that of prostate-specific antigen. Western blotting of benign and malignant tissue homogenates revealed a predominant 58-kd anti-thrombin immunoreactive component. In vitro, anti-thrombin formed complexes more readily with human kallikrein 2, particularly in the presence of heparin, and less efficiently with prostate-specific antigen. Both complexes could be recognized by polyclonal and monoclonal IgGs against anti-thrombin. We conclude that anti-thrombin is widely expressed in prostate cancer but is gradually lost in tumors of high Gleason grade. Anti-thrombin may act as a local anti-angiogenic factor, the effect of which is partially lost in poorly differentiated prostatic tumors.

Animals↗
PubMed1998

Thrombin Generation in Patients with Acute Myocardial Infarction Treated with Front-Loaded rt-PA and Recombinant Hirudin (HBW 023).

Thrombin contributes to the pathogenesis of acute myocardial infarction and reocclusion after thrombolysis. Thrombolytic therapy is known to induce a paradoxic increase in thrombin generation. Specific thrombin inhibition enhances thrombolytic therapy in experimental models. The aim of this study was to determine thrombin generation in patients with acute myocardial infarction treated with rt-PA and conjunctive therapy with the specific thrombin inhibitor, recombinant hirudin. Thrombin generation was determined in 17 patients with acute myocardial infarction treated with front-loaded rt-PA (100 mg/90 min) and conjunctive therapy with recombinant hirudin (HBW 023 bolus 0.4 mg/kg, infusion of 0.15 mg/kg/h) over 48 hours. Mean free hirudin plasma levels of 1320-1545 ng/mL produced a stable anticoagulation with mean aPTT values between 63 and 81 seconds throughout the treatment period. Thrombin generation increased during thrombolysis, indicated by a transient elevation of prothrombin fragment 1.2 levels, which were 3.0 nmol/L at baseline, 11.1 nmol/L after 30 minutes, 8.3 nmol/L after 60 minutes, 3.1 nmol/L after 12 hours, and 1.5 nmol/L after 24 hours, respectively. In contrast, thrombin-antithrombin III complex levels during and after thrombolysis did not exceed the baseline level of 21.8 ug/L. Thrombin-hirudin complex levels increased constantly during the 48-hour treatment period from 3.1 ug/L at baseline to 64.2 ug/L. All patients had an open infarct vessel (TIMI 2/3 potency) after 36-48 hours. Thrombolysis with rt-PA is associated with a significant increase in thrombin generation, which is not blocked by r-hirudin, whereas circulating thrombin seems to be effectively inhibited by r-hirudin.

Journal Article↗
PubMed2005

Determination of prothombinase activation after adding human purified prothrombin to human clot: comparison of hirudin, an activated factor II inhibitor, with DX9065a, an activated factor X inhibitor, on clot-associated thrombin and on prothrombin activation.

Clot-associated prothrombinase and thrombin activities may contribute to thrombus extension after thrombolytic and anticoagulant treatment. We studied prothrombin activation after adding human purified prothrombin to human clot. By using two different drugs with an exclusive direct anti-activated factor X activity (DX9065a) or anti-activated factor II activity (r-hirudin), we tried to determine whether clot-bound thrombin and prothrombinase could be inhibited in our experimental system when human purified prothrombin was added. Standard clots were prepared from platelet-poor human plasma after addition of calcium. We measured clot-bound thrombin or free thrombin using a direct simple chromogenic assay. In parallel, prothrombin fragment 1+2 measurement was used to monitor prothrombin activation. For this, two protocols were used. We introduced the direct inhibitors before starting the activation process (protocol A) or at the time of the activation process (protocol B). We found a direct correlation between thrombin generation and prothrombin fragment 1+2 with an increase of thrombin activity on clots and in the incubation mixtures when clots were incubated in human purified pothrombin alone. Two protocols were used: in the first, clots were pre-incubated in presence of drugs before adding prothrombin; and in the second, clots were incubated in the presence of prothrombin and drugs. Prothrombin activation was not inhibited when clots were incubated with r-hirudin and consequently thrombin generation still occurred. However, added r-hirudin blocks thrombin activity on the clots and in the incubation mixture, but does not prevent prothrombin activation, as shown by the increase of prothrombin fragment 1+2. In contrast, DX9065a did not suppress clot-bound thrombin. However, DX9065a blocks prothrombin activation whichever protocol was used. The results show that hirudin is a poor inhibitor of thrombin generation in contrast to DX9065a. On the other hand, DX9065a cannot inhibit thrombin bound to clot in contrast to hirudin.

Blood Coagulation↗
PubMed2003

Non-peptidic small-molecule antagonists of the human platelet thrombin receptor PAR-1.

The thrombin receptor on human platelets is the first member identified of a new family of G-protein coupled receptors referred to as protease activated receptors (PARs). These receptors are activated by a unique mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. The hexapeptide SFLLRN-NH2 comprising the new N-terminus is referred to as the Thrombin Receptor Activating Peptide, or "TRAP" Thrombin is the most potent agonist for platelet aggregation and selective blockade of the intramolecular activation step without effecting the proteolytic activity of thrombin should result in moderation of platelet activation and aggregation without interfering with the other coagulation cascade effects of thrombin. Screening of combinatorial libraries identified a novel, non-peptide PAR-1 thrombin receptor antagonist. Examination of structure-activity relationships revealed that portions of the molecule could be replaced resulting in simpler molecules of lower molecular weight that were at the same time more potent. Molecules in this series were effective antagonists of TRAP-stimulated platelet activation, but had limited activity when thrombin was the agonist. Additional directed screening and subsequent lead refinement resulted in a second series of isoxazole based compounds. Some of the resultant molecules were potent PAR-1 antagonists that were effective against both TRAP- and thrombin-stimulated receptor activation. These compounds do not inhibit the proteolytic effects of thrombin but rather interfere with the intramolecular binding of the tethered ligand (SFLLRN) to the transmembrane portion of the thrombin receptor. They represent promising leads for future explorations of antithrombotic activity of thrombin receptor antagonists.

Blood Platelets↗
PubMed2000

Meizothrombin, an intermediate of prothrombin activation, stimulates human glioblastoma cells by interaction with PAR-1-type thrombin receptors.

Thrombin induces well-characterized effects on normal and neoplastic brain cells by interaction with protease-activated receptor (PAR)-type thrombin receptors. However, nothing is known about the function of intermediate enzymes of prothrombin activation recently shown to evoke PAR-1-mediated signaling in smooth muscle cells. Therefore, we investigated the effect of recombinant human meizothrombin (rMT), one of thrombin's catalytically active precursor enzymes in the prothrombin cleavage cascade, on calcium mobilization in human SNB-19 glioblastoma cells. By using reverse-transcription polymerase chain reaction, immunofluorescence studies with a monoclonal anti-PAR-1 antibody and calcium measurements, SNB-19 cells were shown to express functional PAR-1-type thrombin receptors. PAR-1 is not only a receptor for thrombin in SNB-19 cells but was also activated by rMT very effectively. Under the conditions used in our experiments, SNB-19 cells stimulated with thrombin after rMT challenge were unable to elicit a new calcium response and vice versa. In addition, both rMT and thrombin induced no further calcium signal after that observed with the PAR-1-activating peptide SFLLRN. Therefore, rMT and thrombin seem to activate calcium signaling by similar mechanisms including PAR-1. Our results demonstrate rMT as a potent activator of PAR-1-type thrombin receptors in SNB-19 glioblastoma cells, suggesting a function of catalytically active thrombin precursor enzymes in cells of glial origin.

Calcium↗
PubMed2003

Thrombin downregulates muscle acetylcholine receptors via an IP3 signaling pathway by activating its G-protein-coupled protease-activated receptor-1.

Regulation of thrombin activity may be required during skeletal muscle differentiation since the thrombin tissue inhibitor protease nexin-1 appears at the myotube stage before being localized at the neuromuscular synapse. Here, we have used a model of rat fetal myotube primary cultures to study the effect of thrombin on acetylcholine receptor (AChR) expression, which is enhanced at the myotube stage. Our results show that thrombin decreases both the number of surface AChRs (AChRn) and AChR alpha-subunit gene expression. Using the agonist peptide SFLLRN, we establish that the AChRn decrease is mediated by the G protein-coupled thrombin receptor "protease-activated receptor-1" (PAR-1). Moreover, the specific thrombin inhibitor hirudin increases AChRn by inhibiting the thrombin intrinsically present in the cultures. We further demonstrate that the activation of PAR-1 by thrombin induces intracellular calcium movements that are blocked by 2-APB, an inhibitor of inositol 1,4,5-triphosphate (IP3)-induced calcium release. These calcium signals are more intense in nuclei than in the cytoplasm and are consistent with the intracellular distribution of IP3 receptor that we find in the cytoplasm in a cross-striated pattern and at a high level in the nuclear envelope zone. Finally, we show that the blockade of these IP3-induced calcium signals by 2-APB prevents the AChRn decrease induced by thrombin. Our results thus demonstrate that thrombin downregulates AChR expression by activating PAR-1 and that this effect is mediated via an IP3 signaling pathway.

Acetylcholine↗
PubMed1994

Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture.

Astrocytes appear star-shaped in the brain, increasingly so after injury. When astroglia are cultured in serum-containing medium, they exhibit a flat, fibroblast-like morphology. In serum-free medium, astrocytes become stellate, with many long processes. The serine protease alpha-thrombin mimics the effects of serum at subnanomolar concentrations, whereas the thrombin-inhibiting serpin, protease nexin I (PNI), reverses the thrombin effect. In our current experiments, murine neonatal spinal cord astrocytes became stellate after 4 hr in serum-free medium, while cortical astrocytes required 12 hr in serum-free medium for stellation. Astrocytes from either region flattened after 60 min in medium containing 3.0 to 300 pM proteolytically active human alpha-thrombin. After 12 hr in thrombin-containing medium, 98% of the astrocytes had a flattened morphology. No flattening occurred if alpha-thrombin was replaced by gamma-thrombin, which has its fibrinogen-recognition exosite disrupted. PNI added at 1 nM to serum-containing medium caused stellation after 3 hr, and astroglia were 50% stellate by 12 hr. The effect of thrombin was mimicked by a 7-amino acid peptide (TRP-7) from the cleavage site of the human thrombin receptor. This peptide caused 40% of the astrocytes in serum-free medium to exhibit a flattened morphology after 6 hr. PNI had no effect on TRP-7 action on astrocytes. These results indicate that astrocytes possess a cell-surface receptor for thrombin, similar to that described for platelets, endothelial cells, and neurons.

Amino Acid Sequence↗
PubMed1994

Thrombin stimulates fibroblast-mediated collagen lattice contraction by its proteolytically activated receptor.

Fibroblast contraction is proposed to play an important role in tissue contraction during events such as wound healing. Thrombin has been implicated to promote force generation in fibroblasts; however, its extracellular mode of action is unclear. The purpose of this study was to determine the role thrombin and the activation of its receptor plays in promoting the contraction of human fibroblasts in an in vitro collagen lattice contraction assay. Human alpha-thrombin promoted fibroblast contraction in a dose-dependent manner with maximal activity at 0.2 nM. In contrast, both hirudin-alpha-thrombin and D-phenylalanyl-L-propyl-L-arginyl chloromethyl ketone-alpha-thrombin, which lack enzymatic activity, failed to elicit fibroblast contraction. Thus, the enzymatic activity of thrombin appears to be necessary for promotion of fibroblast contraction. Northern analysis confirmed that these human fibroblasts expressed mRNA for the human alpha-thrombin receptor. Moreover, the synthetic peptide (SFLLRNPND-KYEPF) representing the "tethered ligand" portion of the activated alpha-thrombin receptor promoted fibroblast contraction, while a control isomer peptide, in which the first two amino acids were reversed, failed to elicit this response. These findings indicate that alpha-thrombin promotes the contraction of adult human fibroblasts and that cleavage of the human alpha-thrombin receptor is sufficient to produce this response.

Amino Acid Chloromethyl Ketones↗
PubMed1998

The thrombin receptor elevates intracellular calcium in adult rat ventricular myocytes.

While there is evidence that thrombin receptor activation leads to contractile dysfunction and induces arrhythmias in ischemic/reperfused cardiac tissue, thrombin is variably reported to modulate intracellular calcium in cardiomyocytes. The present study demonstrates that thrombin receptor activation leads to a rise in intracellular calcium in adult ventricular myocytes and serves to reconcile previous discrepant findings. The thrombin receptor-derived agonist peptide (SFLLRN, a portion of the tethered ligand created by thrombin's proteolytic actions) increases cytosolic calcium and twitch amplitude in cardiomyocytes isolated from adult ventricles. The truncated control peptide FLLRN has no effect, establishing that the response to SFLLRN results from a specific agonist peptide-receptor interaction. However, the response to SFLLRN occurs only at high agonist peptide concentrations and thrombin itself is inactive. This result is not compatible with an action of SFLLRN at a distinct protease-activated receptor (PAR-2; which is activated by SFLLRN, but not by thrombin), since SLIGRL (a ligand which is selective for PAR-2, but not the thrombin receptor) has no effect. Rather, the enzyme-based cell isolation procedure may partially cleave the thrombin receptor and influence cell responses, since concentrations of SFLLRN which are sub-threshold in enzymatically disaggregated myocytes significantly increase the force of isometric contraction of intact rat papillary muscles. These studies provide the first evidence that thrombin receptor activation leads to a change in intracellular calcium and a positive inotropic response in adult ventricular myocardium.

Animals↗
PubMed1996

Regulation of the thrombin receptor response in human endothelial cells.

At present only little information is available about the regulation of the thrombin receptor activity in human endothelial cells. The study presented was performed to clarify the problem of thrombin receptor regulation in human endothelial cells. Endothelial cells were isolated from the vein or artery of human umbilical cords. These cells were stimulated with thrombin or with the thrombin receptor activation peptides (TRAP) SFLLRN or SFLLRNPNDKYEPF and subsequently the von Willebrand factor (vWf) release was measured as a physiologically significant response regarding the thrombin receptor activation. Shortly after the first activation by thrombin or SFLLRN, the cells were desensitised to a second stimulation. After a recovery phase of 10 min, merely 35% of the receptor response was obtained by subsequent stimulation. Expanding the recovery time to 90 min resulted in a vWf release of nearly 100% of the amount measured after the first stimulation. The resensitisation rate was similar for thrombin and SFLLRN stimulated cells. Adding RNA synthesis inhibitors or protein synthesis inhibitors had no effect on the recovery of the receptor response. Therefore, a de novo synthesis of receptor protein must be excluded as a means of resensitising endothelial cells to thrombin. It was shown that the dephosphorylation of tyrosine kinase inactivated receptors is also not responsible for receptor regeneration. These results prove that endothelial cells are capable of developing full thrombin receptor activity after a relatively short recovery phase of only 60-90 min as compared to the recovery phase of 16-24 h in megacaryoblastic cell lines. Desensitisation is similar in thrombin and TRAP stimulated cells. We assumed a transport mechanism for intracellular stored vesicles for receptor resensitisation; a co-localisation with vWf in the Weibel-Palade bodies is improbable.

Amino Acid Sequence↗
PubMed1992

Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Equilibrium binding of thrombin to recombinant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues.

Thrombomodulin is an endothelial cell surface receptor for thrombin that acts as a physiological anticoagulant. The properties of recombinant human thrombomodulin were studied in COS-7, CHO, CV-1, and K562 cell lines. Thrombomodulin was expressed on the cell surface as shown by the acquisition of thrombin-dependent protein C activation. Like native thrombomodulin, recombinant thrombomodulin contained N-linked oligosaccharides, had Mr approximately 100,000, and was inhibited or immunoprecipitated by anti-thrombomodulin antibodies. Binding studies demonstrated that nonrecombinant thrombomodulin expressed by A549 carcinoma cells and recombinant thrombomodulin expressed by CV-1 and K562 cells had similar Kd's for thrombin of 1.3 nM, 3.3 nM, and 4.7 nM, respectively. The Kd for DIP-thrombin binding to recombinant thrombomodulin on CV-1(18A) cells was identical with that of thrombin. Increasing concentrations of hirudin or fibrinogen progressively inhibited the binding of 125I-DIP-thrombin, while factor Va did not inhibit binding. Three synthetic peptides were tested for ability to inhibit DIP-thrombin binding. Both the hirudin peptide Hir53-64 and the thrombomodulin fifth-EGF-domain peptide Tm426-444 displaced DIP-thrombin from thrombomodulin, but the factor V peptide FacV30-43 which is similar in composition and charge to Hir53-64 showed no binding inhibition. The data exclude the significant formation of a ternary complex consisting of thrombin, thrombomodulin, and hirudin. These studies are consistent with a model in which thrombomodulin, hirudin, and fibrinogen compete for binding to DIP-thrombin at the same site.

Amino Acid Sequence↗
PubMed2000

PAR3 is a cofactor for PAR4 activation by thrombin.

Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptor's acting as an accessory molecule to present ligand to another receptor.

Amino Acid Sequence↗