Receptors of the PAR Family as a Link between Blood Coagulation and Inflammation

Blood coagulation plays a key role among numerous mediating systems that are activated in inflammation. Receptors of the PAR family serve as sensors of serine proteinases of the blood clotting system in the target cells involved in inflammation.Activation of PAR-1 by thrombin and of PAR-2 by factor Xa leads to a rapid expression and exposure on the membrane of endothelial cells of both adhesive proteins that mediate an acute inflammatory reaction and of the tissue factor that initiates the blood coagulation cascade. Certain other receptors (EPR-1, thrombomodulin, etc.), which can modulate responses of the cells activated by proteinases through PAR receptors, are also involved in the association of coagulation and inflammation together with the receptors of the PAR family. The presence of PAR receptors on mast cells is responsible for their reactivity to thrombin and factor Xa and defines their contribution to the association of inflammation and blood clotting processes.     

Tissue factor mediates inflammation

The role of tissue factor (TF) in inflammation is mediated by blood coagulation. TF initiates the extrinsic blood coagulation that proceeds as an extracellular signaling cascade by a series of active serine proteases: FVIIa, FXa, and thrombin (FIIa) for fibrin clot production in the presence of phospholipids and Ca2+. TF upregulation resulting from its enhanced exposure to clotting factor FVII/FVIIa often manifests not only hypercoagulable but also inflammatory state. Coagulant mediators (FVIIa, FXa, and FIIa) are proinflammatory, which are largely transmitted by protease-activated receptors (PAR) to elicit inflammation including the expression of tissue necrosis factor, interleukins, adhesion molecules (MCP-1, ICAM-1, VCAM-1, selectins, etc.), and growth factors (VEGF, PDGF, bFGF, etc.). In addition, fibrin, and its fragments are also able to promote inflammation. In the event of TF hypercoagulability accompanied by the elevations in clotting signals including fibrin overproduction, the inflammatory consequence could be enormous. Antagonism to coagulation-dependent inflammation includes (1) TF downregulation, (2) anti-coagulation, and (3) PAR blockade. TF downregulation and anti-coagulation prevent and limit the proceeding of coagulation cascade in the generation of proinflammatory coagulant signals, while PAR antagonists block the transmission of such signals. These approaches are of significance in interrupting the coagulation-inflammation cycle in contribution to not only anti-inflammation but also anti-thrombosis for cardioprotection.     

A novel approach in potential anticoagulants from peptides epitope 558–565 of A2 subunit of factor VIII

Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser⁵⁵⁸–Gln⁵⁶⁵ region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa–FIXa interaction. Based on this, a series of linear peptides, analogs of the 558–565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.     

Coagulation, hemostasis, and plasma expanders:a quarter century enigma.

Despite more than 2 decades of research, the explanation of the long-known hemostatic failure consequent to the use of some natural and synthetic macromolecular agents as plasma substitutes remains obscure. Conventional clotting parameters are not significantly affected in vivo or in vitro. Dextran, hydroxyethyl starch, and many other colloid macromolecules precipitate Factors I and VIII, fibrin monomer, and perhaps v. W. (von Willebrand) factor(s) from plasma, rendering at least the first three insoluble, in relation to the molecule size and concentration of the colloid, and for dextran, its intrinsic viscosity. The precipitate, rich in Factors VIII and I, redissolves on warming, and reprecipitates on cooling, behaving as a cryo-Factor I. In composition it closely resembles the cryoprecipitate obtained by slow-thawing of plasma. Both clot faster with thrombin than the parent plasma. The amount precipitated from plasma by dextran or hydroxyethyl starch varies very widely from individual to individual. Cryo- of dextran-precipitable material can be obtained by interacting purified Factor I with a miniscule amount of thrombin. Dextran, hydroxyethyl starch, polyvinyl pyrrolidone, some forms of gelatin, and several polyamino acids accelerate thrombin clotting of normal plasma, several dysfibrinogenemic plasmas, or Factor I. Albumin, hemoglobin, some modified gelatins do not. Poor platelet thromboplastic function appears some hours after dextran infusion, associated with morphologic capillary abnormalities that strikingly resemble those in v. W. disease. We postulate that the hemostatic defect associated with the use of plasma substitutes is a form of induced v. W. disease or disseminated intravascular clotting, ensuing from precipitation and removal of v. W. factor(s), Factors VIII and I, microcirculatory abnormality, and platelet malfunction. The latter two supervene some time after administration of dextran. It reported antithrombotic activity is perhaps referable to the same action.     

Cyclic peptide analogs of 558–565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants

Novel anticoagulant therapies target specific clotting factors in blood coagulation cascade. Inhibition of the blood coagulation through Factor VIII–Factor IX interaction represents an attractive approach for the treatment and prevention of diseases caused by thrombosis. Our research efforts are continued by the synthesis and biological evaluation of cyclic, head to tail peptides, analogs of the 558–565 sequence of the A2 subunit of FVIII, aiming at the efficient inhibition of Factor VIIIa–Factor IXa interaction. The analogs were synthesized on solid phase using the acid labile 2-chlorotrityl chloride resin, while their anticoagulant activities were examined in vitro by monitoring activated partial thromboplastin time and the inhibition of Factor VIII activity. The results reveal that these peptides provide bases for the development of new anticoagulant agents.     

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR1). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR1-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR1-specific agonists and inhibitors were used to demonstrate that PAR1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR1 and not PAR2. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

PAR1-mediated RhoA activation facilitates CCL2-induced chemotaxis in PC-3 cells

Patients with advanced prostate cancer often exhibit increased activation of the coagulation system. The key activator of the coagulation cascade is the serine protease thrombin which is capable of eliciting numerous cellular responses. We previously reported that the thrombin receptor PAR1 is overexpressed in prostate cancer. To investigate further the role of PAR1 in prostate cancer metastasis, we examined the effects of thrombin activation on cell adhesion and motility in PC-3 prostate cancer cells. Activation of PAR1-induced dynamic cytoskeletal reorganization and reduced PC-3 binding to collagen I, collagen IV, and laminin (P < 0.01) but not fibronectin. Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC-3 cells may be modulating cell adhesion through integrin function but not expression. Furthermore, RhoA was activated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and induced migration towards monocyte chemoattractant protein 1 (MCP-1; CCL2). Thus, it appears that thrombin stimulation plays a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix and positioning the cell in a "ready state" for migration in response to a chemotactic signal. Further exploration is needed to determine whether PAR1 activation affects other signaling pathways involved in prostate cancer.     

The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Effects of high-molecular-weight cryoprotectants on platelets and the coagulation system

The objective of this study is to examine the effects of the most widely used high-molecular-weight cryoprotectants on the coagulation system. Dextran, hydryoxyethyl starch (HES), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), and albumin were added at different concentrations in the range between 0.01-1% (w/v) to solvent/detergent-treated plasma. Using a STA/STA Compact coagulation analyzer the following clotting tests were performed: prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Factor V, and Factor VIII percentage of activity. PVP and PEG caused a significant increase in APTT, a decrease in Factor VIII percentage of activity, and a slight decrease in TT, while PT and Factor V percentage of activity remained unchanged. Dextran, HES, and albumin did not effect the clotting tests. The effect of high-molecular-weight cryoprotectants on platelets was assessed by platelet-induced clot retraction (PICR) and aggregation with thrombin and agglutination with ristocetin. Platelet aggregation and agglutination were unaffected by all cryoprotectants tested; however, PICR was significantly reduced in the presence of PVP or PEG. Possible mechanisms by which PVP and PEG interfere with the coagulation system are discussed. We also raise issues concerning the development of one-step blood cryopreservation techniques which do not require cryoprotectant removal prior to transfusion.     

Evaluation of the efficacy and safety of rivaroxaban using a computer model for blood coagulation

Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Union and several other countries for the prevention of venous thromboembolism in adult patients undergoing elective hip or knee replacement surgery and is in advanced clinical development for the treatment of thromboembolic disorders. Its mechanism of action is antithrombin independent and differs from that of other anticoagulants, such as warfarin (a vitamin K antagonist), enoxaparin (an indirect thrombin/Factor Xa inhibitor) and dabigatran (a direct thrombin inhibitor). A blood coagulation computer model has been developed, based on several published models and preclinical and clinical data. Unlike previous models, the current model takes into account both the intrinsic and extrinsic pathways of the coagulation cascade, and possesses some unique features, including a blood flow component and a portfolio of drug action mechanisms. This study aimed to use the model to compare the mechanism of action of rivaroxaban with that of warfarin, and to evaluate the efficacy and safety of different rivaroxaban doses with other anticoagulants included in the model. Rather than reproducing known standard clinical measurements, such as the prothrombin time and activated partial thromboplastin time clotting tests, the anticoagulant benchmarking was based on a simulation of physiologically plausible clotting scenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivity for tissue factor concentration inducing clotting, and a steep concentration-effect relationship, rapidly flattening towards higher inhibitor concentrations, both suggesting a broad therapeutic window. The predicted dosing window is highly accordant with the final dose recommendation based upon extensive clinical studies.     

The effect of cyanate on the clotting proteins and platelet function.

Cyanate reacts with unchanged amino groups of various proteins in a specific irreversible carbamylation reaction. The effect of this molecule on the clotting process and the effects of carbamylation on the clotting proteins and platelet functions were investigated in vitro. An immediate effect on the clotting proteins, not related to pH, was seen in the screening tests prothrombin time, partial thromboplastin time and thrombin time at the highest concentration (100 mM), to a lesser degree at the lower concentration (10 mM). These changes reflected decreases of 19 and 36% respectively in Factor V and X activity, an inhibition of 63-75% of Factors VII, IX, X and XI activity, and 80% inhibition of thrombin activity. The inhibitory changes of carbamylation increased with time. No changes were seen in the activity of Factors I and VIII. Platelet function studies revealed no inhibition of Factor III release; aggregation was abnormal only at high concentrations with epinephrine and collagen induction and partially reversible by resuspension in normal plasma.     

Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII

The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.     

The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin.

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.     

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor.

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.     

Protease-activated receptors 1 and 4 are shut off with distinct kinetics after activation by thrombin

Protease-activated receptors 1 and 4 (PAR1 and PAR4) mediate thrombin signaling in human platelets. Whether these receptors are redundant, interact, or serve only partially overlapping functions is unknown. We report that PAR1 and PAR4 signal with distinct tempos. In transfected fibroblasts, PAR4 triggered substantially more phosphoinositide hydrolysis per activated receptor than PAR1 and was shut off more slowly than PAR1. Shutoff and internalization of PAR1 depends upon phosphorylation of its carboxyl tail upon receptor activation. In contrast to PAR1, phosphorylation of PAR4 was undetectable, and activation-dependent internalization of PAR4 was much slower than that seen for PAR1. Mutation of potential phosphorylation sites in the carboxyl tail of PAR1 enhanced PAR1 signaling, whereas analogous mutations in PAR4 had no effect. Thus PAR4 signaling is shut off less rapidly than PAR1, probably due to differences in receptor phosphorylation. PAR1 and PAR4 also signaled with distinct tempos in platelets. PAR1 triggered a rapid and transient increase in intracellular calcium, whereas PAR4 triggered a more prolonged response. Together, the tempo of these responses accounted for that triggered by thrombin. Thus differences in the rates at which PAR1 and PAR4 are shut off allow thrombin to trigger intracellular signaling with distinct temporal characteristics.     

Coagulation factor V

The coagulation cascade involves sequential enzymatic activations of serine protease zymogens that converge on the generation of thrombin. Factor V (FV) takes part in this process as a component of the prothrombinase complex. Besides its role as procoagulant factor, it is also involved in the physiologic anticoagulant pathway, by participating in the inactivation of activated factor VIII (FVIIIa). Given the dual role of FV, genetic defects in FV gene may result in opposite hemorrhagic or thrombotic phenotypes. This review focuses on the structure, function (procoagulant and anticoagulant), regulation (activation and inactivation) of FV as well as on the genetic defects associated with mutations in the FV gene.