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Author(s): Deanna R. Todd Tzanetos [sup.1], Chang Yu [sup.4], Marta Hernanz-Schulman [sup.5], Frederick E. Barr [sup.2], Nancy J. Brown [sup.3] Author Affiliations: (1) grid.266539.d, 0000000419368438, Department of Pediatrics, Division of [...], Purpose To determine the incidence and clinical and biomarker predictors of perioperative thrombosis in children with single ventricle physiology undergoing staged palliation. Methods Nineteen patients were enrolled and 16 completed the study. Serial ultrasounds of the central venous system were performed to evaluate for thrombus. Plasma antithrombin III, thrombin-antithrombin complex, protein C, protein S, tissue factor pathway inhibitor, plasminogen activator inhibitor-1, tissue plasminogen activator antigen, D-dimer, soluble CD40 ligand, and urinary thromboxane were measured serially before and after surgery. Cardiopulmonary bypass time, aortic cross clamp time, blood product administration, inotrope score, chest tube output, cardiac function by echocardiography, intensive care unit and hospital lengths of stay, and central venous catheter days were recorded. Results The incidence of perioperative thrombus was 31%. Patients who developed a thrombus had poorer preoperative ventricular function (p = 0.03) and longer cardiopulmonary bypass times (p = 0.03) than those who did not develop a thrombus. Preoperative plasma antithrombin III was lower (p = 0.01) and tissue plasminogen activator antigen concentrations were higher (p = 0.02) in patients with a thrombus compared with patients without a thrombus. When measured over time, antithrombin III remained lower (p = 0.002) and tissue plasminogen activator antigen higher (p = 0.005) in those who developed a thrombus compared with those who did not. There were no other statistically significant differences in biomarkers of coagulation between patients with and without thrombosis. Conclusion One-third of patients undergoing palliative surgery for single ventricle physiology develop thrombosis. Decreased ventricular function, low antithrombin III, and increased tissue plasminogen activator may predict those most suitable for randomized clinical trials of anticoagulation.

Toward understanding their redundancies and interactions in hemostasis and thrombosis, we examined the roles of thrombin receptors (protease-activated receptors, PARs) and the ADP receptor P2RY12 (purinergic receptor P2Y G protein-coupled 12) in human and mouse platelets ex vivo and in mouse models. [Par3.sup.-/-] and [Par4.sup.+/-] mouse platelets showed partially decreased responses to thrombin, resembling those in PAR1 antagonist-treated human platelets. [P2ry12.sup.+/-] mouse platelets showed partially decreased responses to ADP, resembling those in clopid0grel-treated human platelets. [Par3.sup.-/-] mice showed nearly complete protection against carotid artery thrombosis caused by low Fe[Cl.sub.3] injury. [Par4.sup.+/-] and [P2ry12.sup.+/-] mice showed partial protection. Increasing Fe[Cl.sub.3] injury abolished such protection; combining partial attenuation of thrombin and ADP signaling, as in [Par3.sup.-/-]: [P2ry12.sup.+/-] mice, restored it. [Par4.sup.-/-] mice, which lack platelet thrombin responses, showed still better protection. Our data suggest that (i) the level of thrombin driving platelet activation and carotid thrombosis was low at low levels of arterial injury and increased along with the contribution of thrombin-independent pathways of platelet activation with increasing levels of injury; (ii) although P2ry12 acts downstream of PARs to amplify platelet responses to thrombin ex vivo, P2ry12 functioned in thrombin/PAR-independent pathways in our in vivo models; and (iii) P2ry12 signaling was more important than PAR signaling in hemostasis models; the converse was noted for arterial thrombosis models. These results make predictions being tested by ongoing human trials and suggest hypotheses for new antithrombotic strategies. vorapaxar | acute coronary syndromes | myocardial infarction stroke | SCH530348 doi/ 10.1073/pnas.1013309107

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease. coagulation | coagulation factor V | coagulation factor XI | feedback activation doi/ 10.1073/pnas.1004741107

The frequently observed deendothelialization of venous coronary bypass gratis prepared using standard methods exposes subendothelial prothrombotic ceils to blood components, thus endangering patients by inducing acute thromboembolic infarction or long-term proliferative stenosis. Our aim was to gain deeper histological and physiological insight into these relations. An intricate network of subendothelial cells, characterized by histological features specific for true pericytes, was detected even in healthy vessels and forms, coupled to the luminal endothelium, a second leaflet of the macrovascular intima. These cells, and particularly those in the venous intima, express enormous concentrations of tissue factor and can recruit additional amounts of up to the 25-fold concentration within 1 h during preincubation with serun (intimal pericytes of venous origin activate 30.71 [+ or -] 4.07 pmol coagulation factor x x [min.sup.-1] x [10.sup.-6] cells: n = 15). Moreover, decoupled from the endothelium, they proliferate rapidly (generation time, 15 [+ or -] 2.1 h, n = 8). Central regions of atherosclerotic plaques, as well as of those of restenosed areas of coronary vein grafts, consist almost completely of these cells. In stark contrast with the prothrombogenicity of the intimal pericytes, intact luminal endothelium recruits high concentrations of thrombomodulin (CD 141) specifically within its intercellular junctions, activates Protein C rapidly (42 [+ or -] 5.1 pmol/min x [10.sup.6] venous endothelial cells at thrombin saturation: n = 15), can thus actively prevent coagulatory processes, and never expresses histologically detectable and functionally active tissue factor. Given this strongly prothrombotic potential of the intimal pericytes and their overshooting growth behavior in endothelium-denuded vascular regions, they may play important roles in the development of atherosclerosis, thrombosis, and saphenous vein graft disease. endothelium: tissue factor: atherosclerosis: thrombosis: coronary bypass operation doi:10.1152/ajpheart.00343.2009

Pulmonary arterial hypertension (PAH) is a progressive disease of excess vasoconstriction and vascular cell proliferation that results in increased pulmonary vascular resistance and right heart failure. We have previously shown (66) that tissue factor expression is increased in the abnormal vessels of patients and rats with PAH. We hypothesized that tissue factor and its downstream mediator, thrombin, would promote migration of endothelial cells (EC) and the vascular pathology of PAH. Immunostaining revealed EC and a fibronectin-enriched matrix within the 'plexiform-like' lesions in a rat model of severe PAH. In a modifed Boyden assay, protease-activated receptor 1 (PAR1; thrombin receptor) stimulation by agonist peptide or thrombin induced pulmonary microvascular EC (PMVEC) migration when the cells were interacting with fibronectin, but not with other extracellular matrix proteins. Thrombin/fibronectin-induced migration was confirmed in wound healing and angiogenesis assays and was abrogated by the PAR1 antagonist SCH79797 and soluble RGD peptide. This fibronectin dependence was unique to PAR1 activation; other EC agonists evaluated did not induce migration on any matrix, and 10% FBS stimulated similar levels of migration on all matrix proteins tested. Thrombin/fibronectin stimulated autophosphorylation of calcium/calmodulin dependent protein kinase II (CaMKII) in PMVEC, and inhibitors of CaMKII blocked thrombin-induced migration on fibronectin, but had no effect on migration induced by 10% FBS. In contrast, EC isolated from the proximal pulmonary artery migrated in response to most agonists independent of the matrix substrate. Our findings illustrate EC heterogeneity in a single tissue and indicate a novel role for CaMKII in mediating EC migration. Because PMVEC have been shown to have impressive proliferative potential, thrombin/fibronectin-stimulated migration of these cells to a site of injured endothelium is a potential mechanism by which thrombin contributes to the development of vascular lesions in PAH. pulmonary hypertension; plexiform lesions; fibronectin doi: 10.1152/ajplung.90598.2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ahj.2008.09.025 Byline: Rahman Shah Abstract: The platelet, once thought to be solely involved in clot formation, is now known to be a key mediator in various other processes such as inflammation, thrombosis, and atherosclerosis. Therefore, antiplatelet agents have become paramount in the prevention and management of various cardiovascular diseases. However, the currently most widely used antiplatelet drugs, aspirin and clopidogrel, have been shown to reduce the risk of serious vascular events only by approximately one quarter. Similarly, oral glycoprotein IIb/IIIa antagonists have been associated with excess mortality, thus restricting the use of parental glycoprotein IIb/IIIa antagonists to the treatment of acute clinical conditions. Thus, for the prevention of cardiovascular diseases, there is still a clinical need for antiplatelet drugs with higher antithrombotic efficacy but with safety profiles that allow for a preventive long-term administration. Thrombin signaling through the protease-activated receptors (PARs) has been shown to influence a wide range of physiologic and pathologic responses in cardiovascular systems. Thus, interference with PARs appears to be a promising strategy to develop new antiplatelet agents with higher efficacy. This review focuses on the cardiovascular actions of PARs that play a role in normal cardiovascular physiology and that are likely to contribute to cardiovascular diseases. Author Affiliation: Department of Cardiovascular Medicine, Baylor College of Medicine, Houston, TX Article History: Received 6 May 2008; Accepted 2 September 2008

The article explains the importance and phenomenon of activation of platelets in a human body. It discusses the importance of platelets in atherothrombosis.

In this study, we tested the hypothesis that factor XI (FXI) activation occurs in plasma following activation of the extrinsic pathway by thrombin-mediated feedback activation. We used two different assays: (i) a direct measurement of activated FXI by ELISA and (ii) a functional assay that follows the activation of the coagulation cascade in the presence or absence of a FXI inhibiting antibody by monitoring thrombin activity. We failed to detect any FXI activation or functional contribution to the activation of the coagulation cascade in platelet poor or platelet-rich plasma, when activation was initiated by thrombin or tissue factor. Additionally, we found that, in the absence of a contact system inhibitor during blood draw, contact activation of FXI can mistakenly appear as thrombinor tissue-factor-dependent activation. Thus, activation of FXI by thrombin in solution or on the surface of activated platelets does not appear to play a significant role in a plasma environment. These results call for reevaluation of the physiological role of the contact activation system in blood coagulation. coagulation | platelets | contact activation

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 ([sup.38][SFNGGPQNTFEEFPLSDIE.sup.56]) and PAR4 ([sup.51]KSSDKPNP [arrow down] [GYPGKFCANDSDTLELPASSQA.sup.81], [arrow down] site of cleavage) have been solved at 2.0 and 3.5 A resolution, respectively. The cleaved form of PAR3, traced in the electron density maps from Gln-44 to Glu-56, makes extensive hydrophobic and electrostatic contacts with exosite I of thrombin through the fragment 47FEEFPLSDIE56. Occupancy of exosite I by PAR3 allosterically changes the conformation of the 60-loop and shifts the position of Trp-60d [approximately equal to] 10 [Angstrom] with a resulting widening of the access to the active site. The PAR4 fragment, traced entirely in the electron density maps except for five C-terminal residues, clamps Trp-60d, Tyr-60a, and the aryl-binding site of thrombin with Pro-56 and Pro-58 at the P2 and P4 positions and engages the primary specificity pocket with Arg-59. The fragment then leaves the active site with Gly-60 and folds into a short helical turn that directs the backbone away from exosite I and over the autolysis loop. The structures demonstrate that thrombin activation of PAR4 may occur with exosite I available to bind cofactor molecules, like the cleaved form of PAR3, whose function is to promote substrate diffusion into the active site by allosterically changing the conformation of the 60-loop. signal transduction | x-ray structure | allostery

Adhesion of platelets to an injured vessel wall and platelet activation are critical events in the formation of a thrombus. Of the agonists involved in platelet activation, thrombin, collagen, and [...]

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.

It is difficult to overstate the medical importance of the serine protease thrombin. Thrombin is involved in many diverse processes, such as cell signaling and memory, but it is the crucial role that it plays in blood coagulation that commands the interest of the medical community. Thrombosis is the most common cause of death in the industrialized world and, whether through venous thromboembolism, myocardial infarction or stroke, ultimately involves the inappropriate activity of thrombin. The number and type of intrinsic and extrinsic natural mechanisms of targeting thrombin that have evolved validate thrombin as an important physiological target, and provide strategies to knock it out. The more we learn about the natural mechanisms that determine thrombin specificity the more likely we are to develop compounds that selectively alter thrombin activity. In this article, we review the natural mechanisms that regulate thrombin activity and novel approaches to inhibit thrombin based on these mechanisms.

Thrombin-stimulated endothelium synthesizes numerous adhesion molecules to recruit leukocytes; however, it is unknown which intracellular pathways are responsible for this event. A recent report from our laboratory has shown that thrombin induces E-selectin expression and that blocking nuclear factor-[kappa]B (NF-[kappa]B) activity partially blocked both E-selectin expression (60%) and leukocyte recruitment. In this study, we systematically assessed the importance of p38 MAPK in thrombin-induced NF-[kappa]B activation and E-selectin-dependent leukocyte recruitment. Thrombin caused phosphorylation of p38 MAPK, its substrate ATF-2, and JNK MAPK, but not ERK MAPK. The p38 MAPK inhibitors, SKF86002 and SB-203580 only reduced ATF-2 activity. We treated human umbilical vein endothelial cells with SKF86002, 1 h before thrombin stimulation, and noted inhibition of NF-[kappa]B mobilization and complete inhibition of leukocyte rolling and adhesion in a laminar flow chamber. Significant inhibition of leukocyte recruitment and E-selectin expression was also observed with SB-203580. SKF86002 did not affect other systems, including tumor necrosis factor-[alpha]-induced E-selectin-dependent leukocyte recruitment. Moreover, thrombin-induced rapid mobilization of P-selectin from Weibel Palade bodies was not p38 MAPK dependent. These data suggest that thrombin induces p38 MAPK activation, which leads to NF-[kappa]B mobilization to the nucleus and causes the upregulation of E-selectin and subsequent leukocyte recruitment. endothelium; selectins; inflammation

We investigated the effects of thrombin on the induction of heat shock proteins (HSP) 70 and 27, and the mechanism behind the induction in aortic smooth muscle A10 cells. Thrombin increased the level of HSP27 but had little effect on the level of HSP70. Thrombin stimulated the accumulation of HSP27 dose dependently between 0.01 and 1 U/ml and cycloheximide reduced the accumulation. Thrombin stimulated an increase in the level of HSP27 mRNA and actinomycin D suppressed the thrombin-increased mRNA level. Thrombin induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The HSP27 accumulation by thrombin was reduced by SB-203580 and PD-169316 but not by SB-202474. SB-203580 and PD-169316 suppressed the thrombin-induced phosphorylation of p38 MAPK. SB-203580 reduced the thrombin-increased level of HSP27 mRNA. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by thrombin. Dissociation was inhibited by SB-203580. Thrombin induced the phosphorylation of HSP27 and the phosphorylation was suppressed by SB-203580. These results indicate that thrombin stimulates not only the dissociation of HSP27 but also the induction of HSP27 via p38 MAPK activation in aortic smooth muscle cells. serine protease; A20 cell; intracellular signaling

In vivo models of airway inflammation suggest that most protein transudation occurs from bronchial microcirculation. However, due to technical limitations in the isolation and culture of bronchial endothelial cells, most studies of lung vascular permeability have focused on pulmonary endothelium. Thus conditions for culture of sheep bronchial artery endothelial cells (BAEC) and bronchial microvascular endothelial cells (BMVEC) were established. The bronchial artery and the mainstem bronchi, stripped of epithelium, were dissected, and endothelial cells were isolated by enzymatic treatment. BAEC and BMVEC demonstrated positive staining for factor VIII-related antigen, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein, and PECAM-1. Radioligand binding studies confirmed equivalent numbers of bradykinin [B.sub.2] receptors on BAEC and BMVEC. Permeability of BAEC and BMVEC was determined after treatment with bradykinin and thrombin by comparing the translocation of FITC-dextran (mol wt 9,500) across confluent monolayers (n = 10-12). Bradykinin caused a maximal increase in permeability in BAEC (165% increase) and BMVEC (144% increase) by 15 min compared with vehicle controls. Thrombin treatment altered BMVEC permeability only, reaching a maximal response at 60 min (109% increase). These results demonstrate bronchial endothelial cell heterogeneity and establish methods to determine intracellular mechanisms contributing to airway disease in relevant cell systems. bradykinin; bronchial microvasculature; fluorescein isothiocyanate-labeled dextran; sheep; thrombin

Research has been conducted on the thrombin-activatable fibrinolysis inhibitor. The effect of plasmin on the activity regulation of this inhibitor has been investigated and the details are discussed.

Research has been conducted on the factor XIII activation by thrombin. The structural features governing the binding and hydrolysis of the factor XIII AP's site-specific mutants in solution have been investigated and are discussed.

Research has been conducted on the haemadin, amino acid thrombin inhibitor. The effect of the ionic strength on the inhibition kinetics of human alpha-thrombin with haemadin has been investigated via the use of the Debye-Huckel theory modification and the results are reported.

Research has been conducted on the bovine thrombin and human factor Xa. The preparation of the peptide-substituted p-guanidinosalicyl cinnamate library and their analysis as inhibitors of the bovine thrombin and human factor Xa artificial mixture are described.

Research has been conducted on the single-chain plasma protein factor V containin carbohydrate. The ability of these carbohydrate moieties to alter the activated protein C (APC)- catalyzed cleavage and inactivation of the factor V and the cofactor resulting from its activation by alpha-thrombin has been investigated and the results are reported.

The importance of coordinated zinc ion for the inactive proenzyme of thrombin-activable fibrinolysis inhibitor activity and for its inactivation is discused. The native enzyme contains zinc in a 1:1 molar ratio and the inactivation is not caused by zinc release. However, proteolysis can irreversibly inactivate the enzyme.

IGFBP-3 binding to endothelial cells inhibits plasmin and thrombin proteolysis. Am J Physiol Endocrinol Metab 282: E52-E58, 2002.--Insulin-like growth factor-binding protein (IGFBP)-3 contains a highly basic COOH-terminal heparin-binding region, the P3 region, which is thought to be important in the binding of IGFBP-3 to endothelial cells. IGFBP-3 and IGFBP-4, and their chimeras IGFBP-[3.sub.4] and IGFBP-[4.sub.3], were treated with plasmin and with thrombin, proteases known to cleave IGFBP-3. IGFBP-3 was highly susceptible to plasmin, whereas IGFBP-4 was less so. Substitution of the P3 region for the P4 region in IGFBP-4 (IGFBP-[4.sub.3]) increased the ability of the protease to digest IGFBP-[4.sub.3]; substitution of the P4 region for the P3 region in IGFBP-3 (IGFBP-[3.sub.4]) decreased the digestion of IGFBP-[3.sub.4]. When [sup.125]I-labeled IGFBP-3 or [sup.125]I-IGFBP-[4.sub.3] was first bound to vascular endothelial cells, subsequent proteolysis by either plasmin or thrombin was substantially inhibited. Proteolysis of [sup.125]I-IGFBP-[3.sub.4] was not inhibited in the presence of endothelial cells. The P3 peptide was cleaved by plasmin but not by thrombin. We conclude that the P3 region is central to proteolysis of IGFBP-3 by plasmin and thrombin, processes which were inhibited by association of IGFBP-3 with endothelial cells. IGFBP proteases

Research has been conducted on the platelet activating factor, a potent lipid autocoid which is synthesized and presented on the endothelial cell surface after the thrombin stimulation. Results indicate that the thrombin stimulation of the human umbilical artery endothelial cells activates membrane-associated Ca (super)2+ -independent phospholipase A (sub)2 leading to the increased lysoplasmalogen production level.

Moulin, Frederic, Bryan L. Copple, Patricia E. Ganey, and Robert A. Roth. Hepatic and extrahepatic factors critical for liver injury during lipopolysaccharide exposure. Am J Physiol Gastrointest Liver Physiol 281: G1423--G1431, 2001.--Bacterial endotoxin [lipopolysaccharide (LPS)] causes liver injury in vivo that is dependent on platelets, neutrophils [polymorphonuclear leukocytes (PMNs)], and several inflammatory mediators, including thrombin. We tested the hypothesis that thrombin contributes to LPS-induced hepatocellular injury through direct interactions with platelets and/or PMNs in vitro. Perfusion of isolated livers from LPS-treated rats with buffer containing thrombin resulted in a significant increase in alanine aminotransferase (ALT) activity in the perfusion medium, indicating hepatocellular damage. This effect was completely abolished by prior depletion of PMNs from the LPS-treated donor rats but not by depletion of platelets, suggesting interaction between thrombin and PMNs in the pathogenesis. Thrombin did not, however, enhance degranulation of rat PMNs in vitro, and it was not directly toxic to isolated rat hepatocytes in the presence of PMNs even after LPS exposure, suggesting that hepatocellular killing by the PMN-thrombin combination requires the intervention of an additional factor(s) within the liver. In livers from naive donors perfused with buffer containing PMNs and LPS, no injury occurred in the absence of thrombin. Addition of thrombin (10 nM) to the medium caused pronounced ALT release. These results indicate that thrombin and PMNs are sufficient extrahepatic requirements for LPS-induced hepatocellular damage in intact liver. endotoxin; coagulation system; perfused liver; inflammation; necrosis; polymorphonuclear leukocyte; platelet; hepatotoxicity; neutrophil; lipopolysaccharide Received 16 December 2000; accepted in final form 9 August 2001

Results support that thrombin's heparin binding site subregions are involved in the glycoprotein Ib-alpha binding to alpha-thrombin, leading to a full platelet activation. A three-dimensional model of the thrombin binding surface region is presented.

Research has been conducted on the ecotin M84R and thrombin. The structure of the ecotin M84R-thrombin complex has been determined in the effort to investigate how thrombin and ecotin form a complex.

Research has been conducted on the peptide inhibitors of human Factor VII. The identification of these peptides has been performed and the results indicate that the affinity has matured from the naive and partially randomized peptide phage libraries.

Research has been conducted on the isolated from the patient with multiple myeloma monoclonal IgG. Results indicate that this IgG binds to exosite II of thrombin, prolongs the thrombin time and alters the hydrolysis kinetics of synthetic peptide substrates.

A thrombin-binding region in the sixth epidermal growth factor (EGF)-like repeat of human thrombomodulin (TM) has been identified using NMR experiments and titration. The binding of thrombin to the TM peptide induces structuring of the TM peptide at the region corresponding to the TM peptide at a region corresponding to the central beta-hairpin of all EGF-like repeats.

A novel thrombin-like enzyme (TLE) from venom of the snake Agkistrodon contortrix contortrix has been purified by affinity chromatophahy and HPLC. The amino acid seqence was determined. Evidence was found for recognition properties different from those of thrombin in the surface near the primary specificity pocket.

The polymerization mechanism has been studied through analysis of the polymerization of B beta A 68 T, which is the recombinant counterpart of fibrinogen Naples, which is known to have cut down on thrombin binding. Decreased lateral aggregation was seen. It is proposed that in polymerization which is normal a subtle E domain conformation change takes place and modulates the lateral aggregation mechanism. The change is between the release of FpA and that of FpB.

Two residues to Ala or Gln were mutated to resolve evidence in conflict about roles of Lys136 and Lys139. Results show Lys136 forms part of the extended heparin binding site of antithrombin that is involved in the binding of full-length heparin chains, while Lys139 is outside that site.

Results show that amino acid substitution at the highly conserved residue W215 of thrombin significantly alters the catalytic activity toward chromogenic and natural substrates. Data also indicate that W215 residue differentially influences thrombin's physiological responses.

The hypothesis that thrombin stimulates contraction of isolated human bronchial rings was tested. The expression of protease-activated receptors (PAR) in cultured human airway smooth muscle (ASM) was analyzed by RT-PCR analysis with specific primers for PARs to characterized the receptors that mediate thrombin effects on human ASM. The results suggest that alpha-thrombin induced contraction of bronchial rings in vitro. In addition, cultured human ASM cells express PAR1 and PAR2 protein.

The role of protease-activated receptor-1 (PAR-1) in various cells and tissues has been studied by using fibroblasts obtained from PAR-1 deficient mice and through the new N-terminal sequence SFLLRN. PAR-1 is a thrombin receptor that is activated when thrombin cleaves its amino-terminal exodomain. Results indicate that thrombin and SFLLRN have no influence on the fibroblasts but showed activity towards ligands after being transfected with human PAR1 cDNA. These findings indicate that PAR1 mediates SFLLRN signaling in thrombin-desensitized PAR-1 expressing cells.

Changes in cytosolic calcium activation and in human pulmonary artery endothelial cells (HPAEC) retraction response was measured to examine the kinetics of endothelial cell surface expression of proteolytically activated thrombin receptor (PAR-1) in HPAEC. The results indicated that endothelial cell surface PAR-1 can be quickly restored even when it is continuously exposed to a low concentration of thrombin. However, cellular PAR-1 pools are depleted by high thrombin concentrations and longer exposure times.