Inhibition Of Thrombin-induced Platelet Aggregation Research Articles

Preferential Inhibition of Fibrin Mediated Thrombosis in Rodent Models By JNJ-9375, a Novel exosite1 Thrombin Inhibitor

We have previously reported an improved therapeutic index (TI) for JNJ-9375 vs apixaban in rat and cynomolgus monkey models of venous thrombosis and bleeding. We have also reported that JNJ-9375 preferentially inhibited the fibrin component of the thrombus without significant impact on the platelet component under low and high shear conditions in a human translational model of thrombosis (Badimon chamber model). We hypothesized that the novel mode of action of JNJ-9375 which by selectively inhibiting exosite 1 substrates from binding to thrombin (such as fibrinogen and PAR-1) may allow for other thrombin interactions at the active site and exosite 2 including platelet activation by PAR-4 and GP1b.In the present studies, we have compared the antithrombotic effects of JNJ-9375, the active site thrombin inhibitor dabigatran and the factor Xa inhibitor apixaban in well characterized and widely used rat AV-shunt and carotid ferric chloride models of venous and arterial thrombosis, respectively. JNJ-9375, dabigatran and apixaban all maximally inhibited thrombus formation in a dose dependent manner in the rat AV-shunt model of venous thrombosis. Significant inhibition of thrombus weights of 50%, 44% and 57% were observed at 0.3 mg/kg, i.v., 0.1 mg/kg, i.v. and 1 mg/kg, i.v. with JNJ-9375, dabigatran and apixaban respectively. Dose dependent prolongation in coagulation parameters were also observed ex-vivo with JNJ-9375 (TT, ECT, aPTT and PT), dabigatran (TT, ECT, aPTT) and apixaban (aPTT and PT). In addition, there were dose dependent increases in drug exposure.In the rat ferric chloride model of arterial thrombosis, JNJ-9375, dabigatran and apixaban administration also resulted in dose dependent inhibition of thrombus formation as assessed by the time to occlusion (TTO) of the carotid artery and area under the curve (AUC) for carotid blood flow. However, the dose required to inhibit arterial thrombosis Vs venous thrombosis varied substantially for the different agents. With JNJ-9375 we only observed a significant inhibition of arterial thrombosis (TTO and AUC) at a dose of 10 mg/kg, i.v. (Vs 0.3 mg/kg in the AV-shunt model) which represents a 33-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). In contrast, we observed a significant inhibition of arterial thrombosis with dabigatran (AUC) and apixaban (TTO and AUC) at doses of 0.3 mg/kg, i.v. and 3 mg/kg, i.v. (Vs 0.1 mg/kg and 1 mg/kg in the AV-shunt model) respectively. These doses of dabigatran and apixaban represent a 3-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). We have also documented dose dependent prolongations in coagulation parameters and drug exposure with all three agents in the these studies. In addition, we performed ex-vivo platelet aggregation in platelet rich plasma with various platelet agonists (ADP, collagen, AA, rat and human thrombin) in those animals dosed with JNJ-9375. As anticipated and consistent with its mechanism of action, JNJ-9375 only inhibited platelet aggregation to thrombin stimulation but not with other platelet agonists confirming the specificity of JNJ-9375. We observed about 50%, >95% and >95% inhibition of thrombin-induced platelet aggregation at 1, 3 and 10 mg/kg, i.v. with JNJ-9375 respectively. However, we only observed inhibition of arterial thrombosis at 10 mg/kg.In summary, we have demonstrated dose dependent inhibition of thrombosis with JNJ-9375, dabigatran and apixaban in venous and arterial model of thrombosis in rats. However, we have observed a larger difference in the dose required for inhibition of arterial Vs venous thrombosis with JNJ-9375 (33-fold) compared to a more modest 3-fold shift observed for dabigatran and apixaban. These findings with JNJ-9375 demonstrate an anticoagulant profile that preferentially inhibits fibrin mediated clot formation with a more modest impact on platelet driven thrombosis in the present models. This differential profile may account in part for the improved TI over apixaban we had reported earlier in animal models. This profile suggests JNJ-9375 which is currently in early clinical development may also be more amenable to dosing in combination with antiplatelet therapy for indications such as acute coronary syndromes and needs to be tested experimentally. DisclosuresChintala:Janssen Research & Development: Employment. Li:Janssen R&D: Employment. Huang Devine:Janssen Research & Development: Employment. Bunce:Janssen R&D: Employment.

Abstract 54: Differential Regulation of NADPH-oxidases 1 and 2 and a Common Syk/PLC/Calcium-dependent ROS Signaling Pathway Mediating Platelet Activation

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. This study investigates how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) mediates different platelet activation pathways. Approach and Results: ROS generation in different platelet activation pathways are mediated differentially by NOX1 and NOX2. NOX1 -/y platelets showed no defects in platelet aggregation and secretion induced by glycoprotein (GP) VI agonists, collagen-related peptide (CRP), but were partially defective in platelet aggregation and secretion induced by low doses of agonists of G protein coupled receptor (GPCR), thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619. In contrast, NOX2 -/- platelets showed significantly defective platelet aggregation and secretion induced by CRP, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1 -/- platelets stimulated with thrombin, but not CRP, whereas NOX2 -/- platelets were defective ROS generation induced by CRP or thrombin. These differential effects of NOX1 and NOX2 is likely due to upstream differential regulation of these different enzymes, as thrombin-stimulated NOX1-/y platelets and CRP-stimulated NOX2-/- platelets similarly showed defective activation of tyrosine kinase Syk, its downstream target phospholipase Cγ (PLCγ) and calcium mobilization, which is mediated by PLC. Furthermore, mitogen-activated protein kinase pathways, which is another important platelet activation pathway was not significantly affected in either NOX1-/y or NOX2-/- platelets. Finally, NOX-/- platelets is defective in mediating arteriolar thrombosis in vivo, although minimally affected tail bleeding time. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways. The differential roles of these enzyme are due to differential upstream regulation. Both NOX isoforms mediates platelet activation via a common ROS-dependent activation Src-PLC-calcium signaling pathway.

Abstract 18972: Safety and Efficacy of Vorapaxar in Secondary Prevention of Atherosclerotic Disease: A Meta-analysis of Randomized Control Trials

Background: A substantial risk of residual cardiovascular events remains despite the current standard antiplatelet therapy in patients with atherosclerotic disease. Protease activating receptor (PAR-1) antagonist Vorapaxar is a novel antiplatelet agent acting by inhibition of thrombin-induced platelet aggregation. Recently, FDA has approved vorapaxar for long term secondary prevention of vascular events in stable patients with a prior MI or PAD, and without history of stroke or transient ischemic attack. However, these recommendations are largely based on a single phase 3 randomized control trial (TRA 2P TIMI-50). Objective: To study the cumulative evidence for vorapaxar use in patients with atherosclerotic cardiovascular disease. Methods: A systematic review of randomized control trials in MEDLINE, EMBASE, EBSCO, CINAHL, Web of Science and Cochrane databases comparing vorapaxar with placebo was performed. Pre-specified efficacy endpoints were all-cause mortality, CV mortality, myocardial infarction, stroke and repeat revascularization. The pre-specified safety endpoint was the composite of TIMI major and minor bleeding. Risk ratios were used as the metric of choice by applying random effects models.. Results: A total of 5 RCTs with 40,630 patients were included. Compared with placebo, vorapaxar led to a significant risk reduction in terms of myocardial infarction [RR 0.93; 95% CI 0.80-0.92] and stroke [RR 0.84; 95% CI 0.72-0.97]. No differences were observed between vorapaxar and placebo with respect to all-cause mortality [RR 0.99; 95% CI 0.90-1.07], cardiovascular mortality [RR 0.93; 95% CI 0.84-1.02], and repeat revascularization [RR 0.96; 95% CI 0.85-1.08]. Vorapaxar was associated with a higher risk of TIMI major and minor bleeding [RR 1.30; 95% CI 1.02-1.65] compared with placebo. There was insignificant (I-squared) heterogeneity between studies. Conclusion: Vorapaxar reduces the risk of ischemic events in patients with atherosclerotic disease at the cost of an increased bleeding risk.

Abstract 19967: Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. However, it remains unclear whether and how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) play roles in different platelet activation pathways. Here we investigated the role of NOX1 and NOX2 in different platelet activation pathways using NOX1 and NOX2 knockout mice. Approach and Results: NOX1-/- platelets showed selective defects in G protein coupled receptor (GPCR)-mediated platelet activation induced by thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619, but was not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2-/- platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1-/- platelets stimulated with thrombin, but not CRP, whereas NOX2-/- platelets showed reduced ROS generation induced by CRP or thrombin. Interestingly, laser-induced arterial thrombosis was impaired in NOX2-/- mice, and in thrombocytopenic mice transfused with NOX2-/- platelets, suggesting an important role for NOX2-dependent platelet ROS production in the laser-induced injury model of thrombosis. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways: NOX1 mediates GPCR-mediated ROS production and platelet activation, whereas NOX2 plays a general role in GPVI- and GPCR-induced ROS production and platelet activation in vitro , and in laser-induced thrombosis in vivo .

Sitagliptin: Anti-platelet effect in diabetes and healthy volunteers

Sitagliptin, a selective dipeptidyl peptidase-4 inhibitor drug is used to treat type-2 diabetes (T2DM). We investigated the anti-platelet activity of sitagliptin in patients with T2DM and in in vitro samples obtained from healthy humans. Patients with T2DM (27 male + 23 female) were selected and followed up before (control) and after treatment with sitagliptin for up to 3 months. Platelets were isolated from the blood of sitagliptin treated patients and controls. Patients with T2DM treated with sitagliptin for 1and 3 months, showed 10 ± 2% and 30 ± 5% inhibition of platelet aggregation, respectively. For the in vitro study, platelets from 10 normal humans (n = 10) were isolated. Platelet aggregation, intracellular free calcium and tyrosine phosphorylation of multiple proteins were measured by aggregometer, spectrofluorometer and western blotting, respectively. Platelets pre-treated with 5 and 10 µg/ml of sitagliptin, showed 25 ± 4% and 40 ± 6% inhibition of thrombin-induced platelet aggregation, respectively. Sitagliptin decreased intracellular free calcium (2.5-fold) and tyrosine phosphorylation of multiple proteins in thrombin-induced platelet activation. Sitagliptin inhibited platelet aggregation in T2DM as well as in healthy humans. Sitagliptin has significant concentration-dependent anti-platelet activity. This activity was due to its inhibitory effect on intracellular free calcium and tyrosine phosphorylation.

PH-Dependent nanostructure based on isoquinoline-cyclodextrin conjugate for thrombosis therapy

The modification of 3S-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (THIQA) with β-cyclodextrin (β-CD) provides an oral antithrombotic agent, 6-(3′S-isoquinoline-3′-carboxylaminoethylamino)-6-deoxy-β-CD (THIQA-β-CD). In aqueous solution THIQA-β-CD undergoes intermolecular inclusion complexation and forms pH-dependent nanostructures. The morphological feature of THIQA-β-CD is a nanocloud consisting of numerous particles that are 5 nm–6 nm in diameter at pH 3.0. The nanocloud switches to a nanorod ranging from 100 nm to 385 nm in length at pH 7.2, then to nanowires of 50 nm to 530 nm in length at pH 10.1. THIQA-β-CD, which has unusual nanostructures, offers enhanced stability in blood. Inhibition of thrombin-induced platelet aggregation in vitro and demonstrated antithrombotic efficacy in vivo. This investigation demonstrated that the modification of THIQA with β-CD is a promising approach for clinical therapy of thrombus disease. The pH-dependent nanostructures of conjugate provide the desired in vivo antithrombotic activity and in vitro stability in blood. From the Clinical EditorThis study a demonstrates that the modification of 3S-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (THIQA) with beta-cyclodextrin, which leads to pH dependent nanostructure formation, is a promising approach for clinical therapy of thrombotic disease.

Bivalirudin Is a Dual Inhibitor of Thrombin and Collagen-Dependent Platelet Activation in Patients Undergoing Percutaneous Coronary Intervention

Bivalirudin, a direct thrombin inhibitor, is a widely used adjunctive therapy in patients undergoing percutaneous intervention (PCI). Thrombin is a highly potent agonist of platelets and activates the protease-activated receptors, PAR1 and PAR4, but it is not known whether bivalirudin exerts antiplatelet effects in PCI patients. We tested the hypothesis that bivalirudin acts as an antiplatelet agent in PCI patients by preventing activation of PARs on the platelet surface. The effect of bivalirudin on platelet function and systemic thrombin levels was assessed in patients undergoing elective PCI. Mean plasma levels of bivalirudin were 2.7±0.5 μmol/L during PCI, which correlated with marked inhibition of thrombin-induced platelet aggregation and significantly inhibited cleavage of PAR1. Unexpectedly, bivalirudin also significantly inhibited collagen-platelet aggregation during PCI. Collagen induced a conversion of the platelet surface to a procoagulant state in a thrombin-dependent manner that was blocked by bivalirudin. Consistent with this result, bivalirudin reduced systemic thrombin levels by >50% during PCI. Termination of the bivalirudin infusion resulted in rapid clearance of the drug with a half-life of 29.3 minutes. Bivalirudin effectively suppresses thrombin-dependent platelet activation via inhibition of PAR1 cleavage and inhibits collagen-induced platelet procoagulant activity as well as systemic thrombin levels in patients undergoing PCI.

γA/γ’ fibrinogen inhibits thrombin-induced platelet aggregation

The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity.

Effect of oversulfated dermatan sulfate derivatives on platelet aggregation

We have investigated the effect on human platelet aggregation of native dermatan sulfate (DS) and three oversulfated DS derivatives with different sulfur contents, and compared it with that of unfractionated heparin. An inhibitory effect on collagen-induced platelet aggregation was observed only with unfractionated heparin at high concentrations, whereas no inhibitory effect was observed when arachidonic acid was used. Heparin was the most potent inhibitor of the thrombin-induced platelet aggregation in platelet-rich plasma (PRP), whereas the oversulfated DS had a higher potency than the native DS. All these glycosaminoglycans (GAGs) also inhibited thrombin-induced aggregation of washed platelets in the presence of antithrombin (AT) or heparin cofactor II (HCII) but not in their absence. Heparin was by far the most potent inhibitor of washed platelet aggregation in the presence of AT, whereas the inhibitory effects of the DS (native or oversulfated) were lower but dependent on the sulfur content. In the presence of HCII, DSb, a slightly oversulfated DS, had the highest inhibitory effect, whereas heparin and DSd, the most oversulfated derivative, had lower potencies in this case. These data suggest that the inhibition of thrombin-induced platelet aggregation by the oversulfated DS derivatives is related to their ability to potentiate thrombin inactivation by AT or HCII. Hence, the oversulfated DS derivatives may not have an effect per se on the inhibition of platelet aggregation. They may constitute a new class of anticoagulants with enhanced anticoagulant effects in comparison with the native DS, but with only minor side-effects of bleeding in comparison with heparin.

Inhibition of thrombin-induced platelet aggregation using human single-chain Fv antibodies specific for TREM-like transcript-1

TREM-like transcript-1 (TLT-1) is a novel platelet membrane receptor, which has been recently characterized in mice. TLT-1 is expressed exclusively in platelets and megakaryocytes, and its expression is dramatically upregulated upon platelet activation, suggesting that it plays a unique role in hemostasis and/or thrombosis. In this study we identified and characterized highly specific human monoclonal antibodies that bind to TLT-1 by screening a naïve library of single chain Fv fragments (scFvs) displayed on filamentous phage (Thomlinson I library). These scFvs detected plate-bound TLT-1, captured soluble TLT-1, and readily reacted with cell-bound TLT-1 on transfectants and primary human platelets. Most importantly, anti-TLT-1 scFvs inhibited thrombin-mediated human platelet aggregation. This inhibition was specific for thrombin-induced aggregation and was reversible with higher doses of agonist. These data are the first to demonstrate a biological role for TLT-1 and its potential as a therapeutic target. The human scFvs isolated in this study may represent novel anti-platelet therapeutic agents.

Effects of extracts and tannins from Arbutus unedo leaves on rat platelet aggregation

Many cardiovascular diseases such as arterial hypertension are associated with an increase in blood platelet activity. Arbutus unedo (Ericaceae) is a medicinal plant reputed to treat arterial hypertension, so the present study was undertaken in order to determine the antiaggregant effect. The crude aqueous extract showed an inhibition of thrombin-induced platelet aggregation (IC50 = 1.8 +/- 0.09 g/L, n = 10). The subsequent extraction of Arbutus unedo leaves by successive solvents showed that the methanol and ethyl acetate extracts accounted for most of the antiaggregant activity (IC50 = 0.7 +/- 0.08, n = 9; 0.6 +/- 0.05; n = 9, respectively). The tannins isolated from the methanol extract exhibited a strong antiplatelet effect (% of inhibition = 75.3 +/- 1.4, n = 8) and may be the major chemical compounds responsible for this action. Our results support the traditional use of this plant in the preventive or therapeutic treatment of platelet aggregation linked to arterial hypertension.

Low Potential for Interactions Between Melagatran/Ximelagatran and Other Drugs, Food, or Alcohol

Vitamin K antagonists including warfarin are associated with numerous interactions with other drugs and foods. In clinical practice, this complicates the task of maintaining plasma levels of warfarin within a narrow therapeutic window and so maximizing protection against thromboembolic events while minimizing the risk of complications, particularly bleeding. In contrast, ximelagatran has a low potential for pharmacokinetic drug:drug and food interactions. There is no significant metabolism of melagatran, and the main route of elimination of melagatran is renal excretion that appears to occur via glomerular filtration. Most importantly, cytochrome P450 isoenzymes that mediate many drug:drug interactions are not involved in the biotransformation of ximelagatran to melagatran. No significant pharmacokinetic interactions have been observed when oral ximelagatran is administered with a range of agents, including diclofenac, diazepam, nifedipine, digoxin, atorvastatin, or amiodarone. The low potential for drug:drug interactions with ximelagatran is also supported by an analysis of the pharmacokinetic data from clinical studies in patients with atrial fibrillation receiving long-term treatment with oral ximelagatran. Increases of mean melagatran area under the curve and maximum plasma concentration ( Cmax) of up to approximately 80% have been observed when ximelagatran is co-administered with the macrolide antibiotics erythromycin or azithromycin, and the mechanism for this interaction is currently under investigation. The bioavailability of melagatran is not altered by co-administration with food or alcohol. The melagatran-induced prolongation of activated partial thromboplastin time (APTT), an ex vivo coagulation time assay used as a measure of thrombin inhibition, is not altered by other drugs [including digoxin, atorvastatin, acetylsalicylic acid (ASA), and amiodarone], food, or alcohol. The effect of melagatran on capillary bleeding time, which is prolonged as a result of the inhibition of thrombin-induced platelet aggregation, is relatively low and additive to the platelet-inhibitory effect of ASA.

A predominant role for cAMP-dependent protein kinase in the cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein and platelet inhibition in humans

AbstractThe vasodilator-stimulated phosphoprotein (VASP) plays an important role in cGMP-induced platelet inhibition. Since VASP is an in vitro substrate for cGMP-dependent protein kinase (PKG), it has been presumed that VASP phosphorylation induced by cGMP is mediated by PKG. Here we show that, in human platelets, phosphorylation of VASP at Ser239 induced by either cGMP analogs or nitric oxide (NO) donor glyco-SNAP1 is inhibited by PKA inhibitors KT5720, PKI, Rp-Br-cAMPS, and H89, but not by PKG inhibitors KT5823 or Rp-pCPT-cGMPS. Unlike human platelets, cGMP analog–induced phosphorylation of VASP in mouse platelets is inhibited by both PKG and PKA inhibitors. Ineffectiveness of PKG inhibitors in inhibiting VASP phosphorylation in human platelets is not due to an inability to inhibit PKG, as these PKG inhibitors but not PKA inhibitors inhibit a different cGMP-induced intracellular signaling event: phosphorylation of extracellular signal–responsive kinase. Furthermore, PKA inhibitors reverse cGMP-induced inhibition of thrombin-induced platelet aggregation, whereas PKG inhibitors further enhance the inhibitory effect of cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of VASP and platelet inhibition in human platelets.

Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation

Introduction: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic–hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting thrombin in the presence of antithrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and thrombin in the presence of heparin cofactor II (HCII). Materials and Methods: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. Results: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the inhibition of thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. Conclusions: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the inhibition of thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different thrombin inhibition mechanism in platelet-rich plasma (PRP).

Fusion proteins with anticoagulant and fibrinolytic properties: functional studies and structural considerations.

In an effort to combine the benefits of fibrinolytics, such as staphylokinase, with those of thrombin inhibitors for the prevention of vessel reocclusion after vascular injury, we have produced several chimeric proteins with plasminogen-activating and thrombin-inhibiting properties. Fusion proteins were constructed consisting of the modules staphylokinase (Sak), the factor Xa cleavage site, and various dipetalin (Dip) domains (H(6)-Sak-Dip-I+II, H(6)-Sak-Dip-I, and H(6)-Sak-Dip-II). Sak stimulates fibrinolysis via activation of plasminogen, whereas dipetalin is a two-domain, Kazal-type inhibitor of thrombin. NMR spectroscopy of the fusion proteins revealed that the molecular structures of the modules are retained in the fusion protein and that no significant interactions occur between the modules in terms of their functionally relevant regions. In enzymatic thrombin inhibition tests and blood coagulation assays (thrombin, prothrombin, and activated partial thromboplastin times), no significant differences in anticoagulant capacity were observed between the fusion protein H(6)-Sak-Dip-I+II and isolated Dip-I+II, even at nanomolar concentrations. Similar results (i.e., the inhibition of thrombin-induced platelet aggregation and the inhibition of thrombin-induced vascular relaxation) were obtained when the cellular thrombin effects were studied. The fusion protein containing Dip-I has less but still significant thrombin inhibitory effects compared with those of H(6)-Sak-Dip-I+II. In contrast, the H(6)-Sak-Dip-II protein failed to inhibit thrombin in each of the assays used. The plasminogen-activating and fibrinolytic activities of the fusion proteins are similar to those of wild-type Sak. The individual dipetalin domains do not activate plasminogen. In conclusion, the fusion protein H(6)-Sak-Dip-I+II is a bifunctional molecule able to activate fibrinolysis via plasminogen activation and inhibit blood coagulation via direct inhibition of thrombin.

Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation.

Activation of endothelial cells and platelet sequestration play major roles in rejection of xenografts. The histopathology of both hyperacute and acute vascular or delayed rejection of vascularized discordant xenografts is characterized by interstitial hemorrhage and intravascular thrombosis. Agents that prevent platelet activation and consequent microthrombus formation have proven beneficial in xenograft rejection but do not fully preclude vascular thrombosis. Recently, several new anti-platelet therapies have undergone extensive clinical testing for atherosclerotic thrombotic vascular disorders; other putative therapies are undergoing pre-clinical evaluation. We have investigated the effect of several of these novel agents on platelet aggregation in baboons in order to screen for future potential in xenograft rejection models. Drugs tested in these experiments were aurintricarboxylic acid (ATA, von Willebrand Factor-GPIb inhibitor), fucoidin (a selectin-inhibitor), 1-benzylimidazole (1-BI, thromboxane synthase antagonist), prostacyclin (PGI2, endothelial stabilizer), heparin (thrombin antagonist), nitroprusside sodium or nicotinamide (NPN or NA, both NO-donors), and eptifibatide (EFT, GPIIb/IIIa receptor antagonist). These were infused intravenously to nine baboons. Coagulation parameters and platelet counts were monitored and baboons were observed for adverse side-effects. The efficacy of these agents in inhibiting platelet aggregation was assayed in a platelet aggregometer. Treatment with ATA and fucoidin resulted in complete inhibition of platelet aggregation but also in major perturbation of coagulation parameters. 1-BI and PGI2 had no effect when administered alone, but in combination resulted in moderate inhibition of aggregation without disturbance in PT or PTT. NPN and NA had no substantive effects on platelet aggregation. Heparin resulted in specific inhibition of thrombin-induced platelet aggregation and, as anticipated, was associated with moderate prolongation of PTT. Importantly, EFT caused complete inhibition of platelet aggregation without changes in coagulation. Platelet counts, fibrinogen levels, and fibrinogen degradation products remained within the normal ranges in all experiments. Although excellent inhibition of platelet activation was obtained with ATA and fucoidin, clinical use may be precluded by concomitant disturbances of coagulation. Combinations of heparin and EFT may prove beneficial in preventing the thrombotic disorders associated with xenograft rejection while maintaining adequate hemostatic responses. These agents are to be evaluated in our pig-to-primate xenotransplantation models.

Human Factor XII Binding to the Glycoprotein Ib-IX-V Complex Inhibits Thrombin-induced Platelet Aggregation

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.

N-Ethylmaleimide inhibition of thrombin-induced platelet aggregation

The data presented in this report show that N-ethylmaleimide (NEM) is a powerful inhibitor of thrombin-induced platelet aggregation. NEM increased guanosine 3′, 5′-cyclic monophosphate (cGMP) and adenosine 3′, 5′-cyclic monophosphate (cAMP) levels in intact cells. The inhibition of cAMP high-affinity phosphodiesterase and cGMP phosphodiesterase was implicated in the elevation of the cyclic nucleotides. NEM dose dependently blocked the thrombin-stimulated, but not the phorbol myristate acetate-dependent phosphorylation of the protein kinase C substrate pleckstrin. Myosin light chain phosphorylation was also inhibited by NEM. In addition, the sulphydryl reagent inhibited Ca 2+ mobilisation induced by thrombin. The data indicate that phospholipase C activation by thrombin is interrupted by NEM at the level of receptor-mediated signal transduction.

Bothrops jararaca snakes produce several bothrojaracin isoforms following an individual pattern

More than one isoform of bothrojaracin (BJC), a potent and specific thrombin inhibitor isolated from Bothrops jararaca venom, has been found in individual venoms collected from adult snakes. Variations in snake venom composition have previously been associated with factors such as age, sex, geographic origin, season of the year and diet. In order to obtain further information concerning individual patterns of expression of BJC isoforms, we have analyzed five individual Bothrops jararaca snake venoms collected at the same time from adult female snakes from the same geographic region. As expected, crude venoms showed a similar migration pattern on SDS-PAGE. BJC was purified using a procedure which includes an affinity chromatography step (PPACK-thrombin Sepharose). A slight variation in the amount of BJC obtained from individual venom samples was noticed. Inhibition of thrombin-induced platelet aggregation as well as migration pattern on SDS-PAGE (under reducing and non-reducing conditions) and isoelectric focusing varied considerably among BJC samples from the five snakes. The amino-terminal sequences (residues 1–34) of individual BJC samples were compared with the sequence deduced from isolated cDNAs encoding α and β chains of BJC. A high degree of homology was detected, although some residues differed from one sample to other. Altogether, data confirmed the heterogeneity found for BJC purified from individual snakes. Thus, the results indicate that: (1) individual specimens of Bothrops jararaca have different patterns of BJC isoform expression; and (2) it seems that genetic factors, at least in part, determine the variability found in BJC production.

HDL3-mediated inhibition of thrombin-induced platelet aggregation and fibrinogen binding occurs via decreased production of phosphoinositide-derived second messengers 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate.

We demonstrate that physiological concentrations of HDL3 inhibit the thrombin-induced platelet fibrinogen binding and aggregation in a time- and concentration-dependent fashion. The underlying mechanism includes HDL3-mediated inhibition of phosphatidylinositol 4,5-bis-phosphate turnover, 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate formation, and intracellular calcium mobilization. The inhibitory effects of HDL3 on inositol 1,4,5-tris-phosphate formation and intracellular calcium mobilization were abolished after covalent modification of HDL3 with dimethylsuberimidate. Furthermore, they could be blocked by calphostin C and bis-indolylmaleimide, 2 highly selective and structurally unrelated protein kinase C inhibitors. However, the inhibitory effects of HDL3 were not blocked by H89, a protein kinase A inhibitor. In addition, HDL3 failed to induce cAMP formation but stimulated the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. We observed a close temporal relationship between the HDL3-mediated inhibition of thrombin-induced inositol 1,4,5-tris-phosphate formation, intracellular calcium mobilization, and fibrinogen binding and the phosphorylation of the protein kinase C 40- to 47-kD major protein substrate. Taken together, these findings indicate that the HDL3-mediated inhibition of thrombin-induced fibrinogen binding and aggregation occurs via inhibition of phosphatidylinositol 4,5-bis-phosphate turnover and formation of 1,2-diacylglycerol and inositol 1,4,5-tris-phosphate. Protein kinase C may be involved in this process.