Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma

Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.     

Contact activation products are new potential biomarkers to evaluate the risk of thrombotic events in systemic lupus erythematosus

Introduction

Patients with systemic lupus erythematosus (SLE) have persistent platelet activation and an increased risk of thrombotic events, which cannot be accounted for by traditional cardiovascular risk factors. Factor (F)XII has a potentially important role in thrombus formation and is triggered by activated platelets. We therefore asked whether the contact system is involved in inflammation and vascular disease (VD) in SLE.

Methods

Fibrin clots were incubated with purified FXII or whole blood, and the activation and regulation of FXII were studied. Plasma from SLE patients with (n = 31) or without (n = 38) previous VD and from matched healthy controls (n = 68) were analyzed for the presence of complexes formed between contact system enzymes and antithrombin (AT) or C1 inhibitor (C1INH) and evaluated with regard to clinical data and laboratory parameters.

Results

Fibrin clots elicited FXII activation and acted as co-factors for AT. In clotting plasma, the levels of FXIIa-AT increased, and FXIIa-C1INH decreased. A similar reciprocal relationship existed in SLE patients. FXIIa-AT was elevated in the SLE patients with a history of VD, while the corresponding levels of factor FXIIa-C1INH were significantly decreased. FXIIa-AT correlated strongly with platelet parameters. The odds ratio for VD among the SLE patients was 8.9 if they had low levels of FXIIa-C1INH, 6.1 for those with high levels of FXIIa-AT, and increased to 23.4 for those with both decreased levels of FXIIa-C1INH and increased levels of FXIIa-AT.

Conclusions

Activation of FXII is elicited by fibrin during thrombotic reactions in vitro and in vivo, and fibrin acts as a heparin-like co-factor and regulates AT. Patients with SLE had altered levels of FXIIa-serpin complexes, supporting that the contact system is involved in this disease. FXIIa-serpin complexes are strongly associated with previous VD in SLE patients, suggesting that these complexes are potential biomarkers for monitoring and assessing the risk of thrombotic events in SLE.     

Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (k_i/k_a = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.     

Acetylated prothrombin as a substrate in the measurement of the procoagulant activity of platelets: elimination of the feedback activation of platelets by thrombin.

Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.     

Ongoing Contact Activation in Patients with Hereditary Angioedema

Hereditary angioedema (HAE) is predominantly caused by a deficiency in C1 esterase inhibitor (C1INH) (HAE-C1INH). C1INH inhibits activated factor XII (FXIIa), activated factor XI (FXIa), and kallikrein. In HAE-C1INH patients the thrombotic risk is not increased even though activation of the contact system is poorly regulated. Therefore, we hypothesized that contact activation preferentially leads to kallikrein formation and less to activation of the coagulation cascade in HAE-C1INH patients. We measured the levels of C1INH in complex with activated contact factors in plasma samples of HAE-C1INH patients (N=30, 17 during remission and 13 during acute attack) and healthy controls (N=10). We did not detect differences in enzyme-inhibitor complexes between samples of controls, patients during remission and patients during an acute attack. Reconstitution with C1INH did not change this result. Next, we determined the potential to form enzyme-inhibitory complexes after complete in vitro activation of the plasma samples with a FXII trigger. In all samples, enzyme-C1INH levels increased after activation even in patients during an acute attack. However, the levels of FXIIa-C1INH, FXIa-C1INH and kallikrein-C1INH were at least 52% lower in samples taken during remission and 70% lower in samples taken during attack compared to samples from controls (p<0.05). Addition of C1INH after activation led to an increase in levels of FXIIa-C1INH and FXIa-C1INH (p<0.05), which were still lower than in controls (p<0.05), while the levels of kallikrein-C1INH did not change. These results are consistent with constitutive activation and attenuated depletion of the contact system and show that the ongoing activation of the contact system, which is present in HAE-C1INH patients both during remission and during acute attacks, is not associated with preferential generation of kallikrein over FXIa.     

Factor XII in coagulation,inflammation and beyond

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe^−/− mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.     

Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor of reaction mechanism

Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor.enzyme template bridging by heparin. Rationales behind this novel concept are discussed.     

The Blood Platelet as a Model for Regulating Blood Coagulation on Cell Surfaces and Its Consequences

Platelets actively participate in regulating thrombin production following physical or chemical injury to blood vessels. Injury to blood vessels initiates activation of the large numbers of platelets that appear in the subendothelium where they become exposed to tissue factor and to molecules adhesive for platelets and normally found in the extracellular matrix. The complex of plasma factor VIIa with extravascular tissue factor both initiates and localizes thrombin production on platelets and on extravascular cells. Thrombin production at these sites in turn enhances platelet activation and the subsequent hemostatic plug formation to minimize bleeding. Thrombin production and platelet activation also initiate the process of wound healing requiring thrombin-dependent cell activation and platelet-dependent formation of new blood vessels (angiogenesis). Activated platelets release from their storage granules several proteins and other factors that regulate local thrombin formation and the responses of blood vessel cells to injury to assure hemostasis and effective wound healing. Failure to localize and adequately regulate thrombin production and/or platelet activation can have pathological consequences, including the development and propagation of atherosclerosis and enhancement of tumor development. The primary basis for the pathological consequences of the failure to adequately regulate thrombin production is that the multi-functional thrombin activates several types of cells to initiate their mitogenesis. Mitogenesis precedes many of the undesirable consequences of poorly regulated thrombin production and platelet activation. In addition, activated platelets release a variety of products which influence the functions of several cell types to the extent that inadequate regulation of platelet activation (by excessive thrombin production) could contribute to the pathogenesis of acute and chronic arterial thrombosis and to tumor development. Activated platelets participate in tumor development by releasing several factors that positively (and negatively) regulate blood vessel formation.     

Factor XI homodimer structure is essential for normal proteolytic activation by factor XIIa, thrombin, and factor XIa

Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because FXI(C321S) is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXI(C321S), FXI(C321S,K331A), FXI(C321S,E287A), FXI(C321S,I290A), FXI(C321S,Y329A), FXI(C321S,L284A), FXI(C321S,K331R), and FXI(C321S,H343A) were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXI(C321S) and FXI(C321S,H343A) existed in monomer/dimer equilibrium (K(d) approximately 40 nm), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K(d)=3-38 microm). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.     

Differential binding of factor XII and activated factor XII to soluble and immobilized fibronectin--localization of the Hep-1/Fib-1 binding site for activated factor XII

Fibronectins (FNs) are dimeric glycoproteins that adopt a globular conformation when present in plasma and solution and an extended conformation in the extracellular matrix. Factor XII (FXII) is a zymogen of the proteolytically active FXIIa that plays a role in thrombus stabilization by enhancing clot formation and in inflammation by enhancing bradykinin formation. To investigate whether the extracellular matrix could play a role in these events, we have recently shown that FXIIa, but not FXII, binds to the extracellular matrix (ECM), and suggested that FN may be the target for the binding. Immunofluorescence microscopy has in the present investigation confirmed that FXIIa added to the ECM colocalizes with FN deposited during growth of human umbilical vein endothelial cells. The aim of the present study, therefore, was to further elucidate the interaction between FXIIa and FN by the use of a solid face binding assay. This showed, like the binding to the ECM, that FXIIa, but not FXII, binds in a Zn2+-independent manner to immobilized FN. The K(D) for the binding was 8.5 +/- 0.9 nM (n = 3). The binding was specific for the immobilized FN, as the binding could not be inhibited by soluble FN. Furthermore, soluble FN did not bind to immobilized FXIIa. However, soluble FN could bind to FXII, and this binding inhibited the surface-induced autoactivation of FXII and subsequent binding of the generated FXIIa to immobilized FN. The presence of FXII in an anti-FN immunoprecipitate of plasma indicated that some FXII in plasma circulates bound to FN. The binding of FXIIa to FN was inhibited by gelatine and fibrin but not by heparin, indicating that FXIIa binds to immobilized FN through the type I repeat modules. Accordingly, FXIIa was found to bind to immobilized fragments of FN containing the type I repeat modules in the N-terminal domain to which fibrin and gelatine bind.     

Binding of activated Factor XII to endothelial cells affects its inactivation by the C1-esterase inhibitor.

It is well known that activated Factor XII (FXIIa) and kallikrein are rapidly inactivated in plasma as a result of reaction with endogenous inhibitors. The purpose of this may be to prevent uncontrolled deleterious spreading and activation of target zymogens. Both FXII and the complex plasma prekallikrein/high molecular mass kininogen become activated when they bind, in a Zn2+-dependent manner, to receptors on human umbilical vein endothelial cells (HUVEC). The C1-esterase inhibitor (C1-INH) is by far the most efficient inhibitor of FXIIa. In the present study it has been investigated whether binding of FXIIa to HUVEC might offer protection against inactivation by C1-INH. It appeared that the relative amidolytic activity of purified FXIIa bound to the surface of HUVEC decreased according to the concentration of C1-INH in medium; however, the decrease was smaller than that measured for inactivation of FXIIa in solution. The secondary rate constant for the inactivation was 3-10-fold lower for cell-bound than for soluble FXIIa. The inactivation was found to be caused by C1-INH binding to cell-bound FXIIa. Accordingly, the amidolytic activity of saturated amounts of cell-bound FXIIa was reduced in the presence of C1-INH and was theoretically nonexistent at physiological C1-INH concentrations. Amidolytic activity was, however, present on HUVEC incubated with plasma indicating that the endogenous C1-INH did not completely abolish the activity of FXIIa generated during the incubation period. This supports the hypothesis that binding to endothelial cells protects the activated FXII against inactivation by its major endogenous inhibitor. Hence, the function of FXII may be localized at cellular surfaces.     

Influence of cysteine proteinase inhibitors on platelet and plasma components of blood coagulation system

Factor XIIIa plays an important role in stabilization of formed fibrin clot during blood coagulation. Recent studies proved that factor XIIIa affects formation of coated platelets, which are highly procoagulant and characterized by a high level of alpha-granular proteins on their surface and expose surface phosphatidylserine after platelet activation. The ability of newly found cysteine proteinase inhibitors (CPIs) from plants to affect thiol group of the factor XIIIa active centre was recently discovered. Here, the effect of CPIs on the formation of coated platelets and activity of plasma components during blood coagulation process was investigated. It was found that CPIs dose-dependently decreased the fraction of coated platelets in the total platelet population during platelet activation and decreased endogenous thrombin potential (ETP) by 40% for thrombin generation in platelet-rich as well as in platelet-poor plasma. Such decrease of ETP could not be explained by the CPIs influence on factor XIIIa. Investigation of the effects of these inhibitors on factor Xa and thrombin activity has shown that CPIs dose-dependently inhibited their activity and might cause an ETP decrease. Thus, the obtained data indicated that CPIs affected both platelet and plasma components of blood coagulation system.     

Development of platelet contractile force as a research and clinical measure of platelet function

This article reviews work performed at the Medical College of Virginia of Virginia Commonwealth University during the development of a whole-blood assay of platelet function. The new assay is capable of assessing platelet function during clotting and thus allows measurement of the contribution of platelets to thrombin generation. Because platelets are monitored in the presence of thrombin, the test gages platelets under conditions of maximal activation. Three parameters are simultaneously assessed on one 700-μL sample of citrated whole blood. Platelet contractile force (PCF), the force produced by platelets during clot retraction, is directly measured as a function of time. This parameter is sensitive to platelet number, platelet metabolic status, glycoprotein IIb/IIIa status, and the presence of antithrombin activities. Clot elastic modulus (CEM), also measured as a function of time, is sensitive to fibrinogen concentration, platelet concentration, the rate of thrombin generation, the flexibility of red cells, and the production of force by platelets. The third parameter, the thrombin generation time (TGT) is determined from the PCF kinetics curve. Because PCF is absolutely thrombin dependent (no thrombin—no force), the initial upswing in PCF occurs at the moment of thrombin production. TGT is sensitive to clotting factor deficiencies, clotting factor inhibitors, and the presence of antithrombins, all of which prolong the TGT and are known to be hemophilic states. Treatment of hemophilic states with hemostatic agents shortens, the TGT toward normal. TGT has been demonstrated to be shorter and PCF to be increased in coronary artery disease, diabetes mellitus, and several other thrombophilic states. Treatment of thrombophilic states with a variety of heparin and nonheparin anticoagulants prolongs the TGT toward normal. The combination of PCF, CEM, and TGT measured on the same sample may allow rapid assessment of global hemostasis and the response to a variety of procoagulant and anticoagulant medications.     

Fibrinolytic properties of activated FXII.

Activated factor XII (FXIIa), the initiator of the contact activation system, has been shown to activate plasminogen in a purified system. However, the quantitative role of FXIIa as a plasminogen activator in contact activation-dependent fibrinolysis in plasma is still unclear. In this study, the plasminogen activator (PA) activity of FXIIa was examined both in a purified system and in a dextran sulfate euglobulin fraction of plasma by measuring fibrinolysis in a fibrin microtiter plate assay. FXIIa was found to have low PA activity in a purified system. Dextran sulfate potentiated the PA activity of FXIIa about sixfold, but had no effect on the PA activity of smaller fragments of FXIIa, missing the binding domain for negatively charged surfaces. The addition of small amounts of factor XII (FXII) to FXII-deficient plasma induced a large increase in contact activation-dependent PA activity, as measured in a dextran sulfate euglobulin fraction, which may be ascribed to FXII-dependent activation of plasminogen activators like prekallikrein. When more FXII was added, PA activity continued to increase but to a lesser extent. In normal plasma, the addition of FXII also resulted in an increase of contact activation-dependent PA activity. These findings suggested a significant contribution of FXIIa as a direct plasminogen activator. Indeed, at least 20% of contact activation-dependent PA activity could be extracted from a dextran sulfate euglobulin fraction prepared from normal plasma by immunodepletion of FXIIa and therefore be ascribed to direct PA activity of FXIIa. PA activity of endogenous FXIIa immunoadsorped from plasma could only be detected in the presence of dextran sulfate. From these results it is concluded that FXIIa can contribute significantly to fibrinolysis as a plasminogen activator in the presence of a potentiating surface.     

Neutrophil cathepsin G promotes prothrombinase and fibrin formation under flow conditions by activating fibrinogen-adherent platelets

Human neutrophil proteases cathepsin G and elastase can directly alter platelet function and/or participate in coagulation cascade reactions on the platelet or neutrophil surface to enhance fibrin formation. The clotting of recalcified platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (to shut down contact activation) was studied in well-plates or flow assays. Inhibitors of cathepsin G or elastase significantly delayed the burst time (t(50)) of thrombin generation in neutrophil-supplemented PRP from 49 min to 59 and 77 min, respectively, in well-plate assays as well as reduced neutrophil-promoted fibrin deposition on fibrinogen-adherent platelets under flow conditions. In flow assays, purified cathepsin G was a far more potent activator of platelet-dependent coagulation than elastase. Anti-tissue factor had no effect on neutrophil protease-enhanced thrombin formation in PRP. The addition of cathepsin G (425 nm) or convulxin (10 nm) to PRP dramatically reduced the t(50) of thrombin generation from 53 min to 17 or 23 min, respectively. In contrast, the addition of elastase to PRP left the t(50) unaltered. Whereas perfusion of PFP (gamma(w) = 62.5 s(-1)) over fibrinogen-adherent platelets did not result in fibrin formation until 50 min, massive fibrin could be observed on cathepsin G-treated platelets even at 35 min. Cathepsin G addition to corn trypsin inhibitor-treated PFP produced little thrombin unless anionic phospholipid was present. However, further activation inhibition studies indicated that cathepsin G enhances fibrin deposition under flow conditions by elevating the activation state of fibrinogen-adherent platelets rather than by cleaving coagulation factors.     

Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces

Human blood coagulation factor XI was activated by either autoactivation or thrombin. These reactions occurred only in the presence of negatively charged materials, such as dextran sulfate (approximately Mr 500,000), sulfatide, and heparin. During the activation, factor XI was cleaved at a single Arg-Ile bond by thrombin or factor XIa to produce an amino-terminal 50-kDa heavy chain and a carboxyl-terminal 35-kDa light chain. This activation pattern is identical to that produced by factor XIIa. The addition of a small amount of thrombin and sulfatide to factor XII-deficient plasma produced shorter clotting times than when these agents were added to factor XI/factor XII combined-deficient plasma. These results suggest that the activation of factor XI by thrombin and possibly the autoactivation of factor XI proceed in plasma to lead fibrin clot formation. These reactions may have a role on an appropriate negatively charged surface in normal hemostasis.     

Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma-thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

FXII

The plasma protein FXII (Hageman factor) has been shown to be linked with the plasma defence systems of coagulation, fibrinolysis, kallikrein-kinin and complement. It can be activated by surface contact activation and in solution. Surface contact activation is a complex phenomenon involving negatively charged surfaces, FXII, high molecular weight kininogen and plasma kallikrein. Fluid-phase activation can be effected by a variety of serine proteases. In both types of activation the FXII zymogen is converted to active enzymes. FXII levels in plasma are low or undetectable in both inherited deficiencies and in a variety of clinical conditions. FXII levels can also be elevated in some clinical conditions. Although discovered as a clotting protein FXII appears to play an important role in the kallikrein-kinin and fibrinolytic systems and also has effects on cells. Recent studies suggest that therapeutic blockade of activation of FXII can be of benefit in certain clinical conditions.     

Antioxidants change platelet responses to various stimulating events

The role of platelets in hemostasis may be influenced by alteration of the platelet redox state—the presence of antioxidants and the formation of reactive oxygen and nitrogen species. We investigated the effects of two antioxidants, resveratrol and trolox, on platelet activation. Trolox and resveratrol inhibited aggregation of washed platelets and platelet-rich plasma activated by ADP, collagen, and thrombin receptor-activating peptide. Resveratrol was a more effective agent in reducing platelet static and dynamic adhesion in comparison with trolox. The antioxidant capacity of resveratrol was, however, the same as that of trolox. After incubation of platelets with antioxidants, the resveratrol intraplatelet concentration was about five times lower than the intracellular concentration of trolox. Although both antioxidants comparably lowered hydroxyl radical and malondialdehyde production in platelets stimulated with collagen, TxB₂ levels were decreased by resveratrol much more effectively than by trolox. Cyclooxygenase 1 was inhibited by resveratrol and not by trolox. Our data indicate that antioxidants, apart from nonspecific redox or radical-quenching mechanisms, inhibit platelet activation also by specific interaction with target proteins. The results also show the importance of studying platelet activation under conditions of real blood flow in contact with reactive surfaces, e.g., using dynamic adhesion experiments.     

Distinct roles for Rap1b protein in platelet secretion and integrin αIIbβ3 outside-in signaling

Rap1b is activated by platelet agonists and plays a critical role in integrin α(IIb)β(3) inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that α(IIb)β(3) outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y(12) or TXA(2) receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl(2), which elicits α(IIb)β(3) outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin α(IIb)β(3) outside-in signaling.