Simone J.H. Wielders

Synergistic Effect of Thrombin on Collagen-Induced Platelet Procoagulant Activity Is Mediated Through Protease-Activated Receptor-1

Objective—In the blood coagulation process, the rate of thrombin formation is critically dependent on phosphatidylserine (PtdSer) at the surface of activated platelets. Thrombin synergistically enhances the collagen-induced platelet procoagulant response. The objective of this study is to elucidate the mechanism of this synergistic action with a focus on the intracellular Ca2+ concentration ([Ca2+]i) and the various platelet receptors for thrombin. Methods and Results—We demonstrate that procoagulant activity is related to a sustained increased [Ca2+]i, which in turn depends on extracellular Ca2+ influx. Increased PtdSer exposure coincides with increased [Ca2+]i and was observed in a subpopulation (≈14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIbα made clear that this receptor did not signal for platelet procoagulant activity. Inhibition of protease-activated receptor 1 (PAR-1) and PAR-4 by selective intracellular inhibitors and selective desensitization of these receptors revealed that PAR-1, but not PAR-4, activation is a prerequisite for both sustained elevations in [Ca2+]i and procoagulant activity induced by collagen plus thrombin. Conclusions—The interaction of thrombin with PAR-1 mediates a synergistic effect on collagen-induced procoagulant activity by inducing a sustained elevation in [Ca2+]i in a subpopulation of platelets.

Thrombogenicity of polysaccharide-coated surfaces.

Heparinization of artificial surfaces has been proven to reduce the intrinsic thrombogenicity of such surfaces. The mechanism by which immobilized heparin reduces thrombogenicity is not completely understood. In the present study heparin-, alginic acid- and chondroitin-6-sulphate-coated surfaces were examined for protein adsorption, platelet adhesion and thrombin generation. The protein-binding capacity from solutions of purified proteins was significantly higher for heparin-coated surfaces when compared with alginic acid- and chondroitin sulphate-coated surfaces. Yet, when the surfaces were exposed to flowing plasma, only the heparinized surface adsorbed significant amounts of antithrombin. None of the surfaces adsorbed fibrinogen under these conditions, and as a result no platelets adhered from flowing whole blood. Our results indicate that protein adsorption and platelet adhesion from anticoagulated blood cannot be used to assess the thrombogenicity of (coated) artificial surfaces. Indeed, the thrombin generation potentials of the different surfaces varied remarkable: while non-coated surface readily produced thrombin, alginic acid- and chondroitin sulphate-coated surfaces showed a marked reduction and virtually no thrombin was generated in flowing whole blood passing by heparinized surfaces.

Factor XI–Dependent Reciprocal Thrombin Generation Consolidates Blood Coagulation when Tissue Factor Is Not Available

Objective—Feedback activation of factor XI by thrombin is a likely alternative for tissue factor-dependent propagation of thrombus formation. However, the hypothesis that thrombin can initiate and propagate its formation in a factor XI-dependent and platelet-dependent manner has not been tested in a plasma milieu. Methods and Results—We investigated thrombin generation in recalcified platelet-rich plasma activated with varying amounts of thrombin or factor VIIa. Thrombin initiates and propagates dose-dependently thrombin generation only when platelets and plasma factor XI are present. Incubation of thrombin-activated platelets with a tissue factor neutralizing antibody had no effect on thrombin formation, indicating that platelet-associated tissue factor, if present at all, is not involved. In the absence of factor VIII, thrombin could not initiate its own formation, whereas factor VIIa-induced thrombin generation was reduced. Collagen strongly stimulated both thrombin-initiated and factor VIIa-initiated thrombin generation. Conclusions—These findings support the notion that platelet-localized feedback activation of factor XI by thrombin plays an important role in maintaining normal hemostasis as well as in sustaining thrombus formation when the TF pathway is inhibited by tissue factor pathway inhibitor.

Covalently-Bound Heparin Makes Collagen Thromboresistant

Objective—Blood compatibility of artificial surfaces depends on their immunogenic and thrombogenic properties. Collagen’s weak antigenicity makes it an attractive candidate for stent coatings or fabrication of vascular grafts. However, the thrombogenic nature of collagen limits its application. We examined whether heparinization can make collagen more thromboresistant. Methods and Results—Collagen was heparinized by crosslinking collagen with extensively periodate oxidized heparin and/or by covalently bonding of mildly periodate oxidized heparin. Both ways of heparinization have no effect on platelet adhesion and could not abolish induction of platelet procoagulant activity. However, thrombin generation was completely prevented under static and flow conditions. The functionality of immobilized heparin was confirmed by specific uptake of antithrombin, 13.5±4.7 pmol/cm2 and 1.95±0.21 pmol/cm2 for mildly and heavily periodated heparin, respectively. Conclusions—These results indicate that immobilization of heparin on collagen, even as a crosslinker, is a very effective way to prevent surface thrombus formation. These data encourage the application of heparinized collagen as stent-graft material in animal and eventually human studies.

Factor Xa Activation of Factor V is of Paramount Importance in Initiating the Coagulation System: Lessons from a Tick Salivary Protein

Background— Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Methods and Results— Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. Conclusions— Our data elucidate a unique molecular mechanism by which ticks inhibit the host’s coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

Anticoagulant and antithrombotic properties of intracellular protease-activated receptor antagonists

Background: Blockade of the thrombin receptors protease‐activated receptor (PAR)1 and PAR4 with pepducins, cell‐penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen‐induced externalization of phosphatidylserine (PS) at the platelet plasma membrane. Objective: In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)‐initiated thrombin generation in platelet‐rich plasma (PRP) and the interaction between PAR4 pepducin‐loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data. Results: Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF‐initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin‐induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus. Conclusions: Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet–platelet interactions.

Prevention of the influence of fibrin and α2-Macroglobulin in the continuous measurement of the thrombin potential : Implications for an endpoint determination of the optical density

We proposed the endogenous thrombin potential (ETP) as an overall function test of the coagulation system. We recently introduced a routine test which requires defibrinated plasma. In order to develop an assay in which the ETP-value can be directly obtained by measuring the optical density, we investigated two methods to inhibit fibrinogen clottability and to inactivate alpha2-macroglobulin. The first method makes use of hydroxylamine to inactivate alpha2-macroglobulin and H-Gly-Pro-Arg-Pro-OH to inhibit fibrin polymerization. At pH 7.35, plasma incubated with 25 mM hydroxylamine and 1.5 mg/mL H-Gly-Pro-Arg-Pro-OH for 5 minutes at 37 degrees C resulted in a reduced endlevel of the amidolytic activity on small chromogenic substrates. The second method uses a metalloprotease purified from Crotalus basiliscus to remove alpha2-macroglobulin from plasma in combination with H-Gly-Pro-Arg-Pro-OH. Herein plasma is incubated with 3.5 LM protease during 15 minutes at 37 degrees C in the presence of 1 mg/mL polymerization inhibitor. The enzymatic method results in a zero endlevel of the amidolytic activity and this would imply that measurement of the ETP is reduced to an endpoint determination of the optical density. We show that the endpoint determination of the optical density correlates well with the calculated ETP in plasmas with different degrees of anticoagulation.

Design and synthesis of thrombin substrates with modified kinetic parameters

For the continuous registration of thrombin formation in plasma (1), selective thrombin substrates are required, that show moderate binding affinities (high Km) and low turnover numbers (low kcat). Previously we have used SQ68 (CH3O-CO-CH2-CO-Aib-Arg-pNA) for this purpose. In order to find more substrates suitable for this application, we synthesized a series of 25 peptide p-nitroanilides. As lead structures SQ68 and S2238 (H-D-Phe-Pip-Arg-pNA) were used. By introduction of specific structure modifications we tried to alter the kinetic data in the required direction. The modifications were designed on basis of existing knowledge on the structure of the thrombin active-site and its surroundings. We indeed obtained a number of substrates with the kinetic constants in the desired range.

Absence of platelet-dependent fibrin formation in a patient with Scott syndrome

To gain insight into the contribution of platelet-dependent thrombin formation in haemostasis and thrombosis, we investigated under flow conditions the haemostatic functions of platelets from a patient with Scott syndrome. Scott platelets are characterised by a diminished platelet-dependent thrombin generation. Thrombin generation was determined by calibrated automated thrombography and flow-based experiments were performed to reveal collagen-mediated platelet activation and fibrin deposition. Our studies indicate that adherent Scott platelets do not differ from control platelets in the formation of stable platelet aggregates under static and flow conditions. While for adherent control platelets a shape change, e.g. balloon formation, and externalisation of phosphatidylserine (PS) is associated with an increase in intracellular calcium concentration, this is not the case for Scott platelets. The calcium-induced morphological changes in control platelets are accompanied with a diminished recruitment of free flowing platelets. Scott platelets, not showing a calcium-induced shape change, also lost the ability to recruit free flowing platelets. These findings rebut the hypothesis that the mild bleeding tendency of Scott syndrome patients is due to a preserved adhesive activity of patients platelets. Perfusion of tissue factor (TF)-activated control blood over immobilised collagen results in the formation of fibrin fibers that radiate from platelet aggregates. Although platelet aggregates were also observed after perfusion with TF-activated Scott blood, fibrin deposition was not observed. In conclusion, our findings indicate that platelet adhesion and spreading on a collagen matrix in the absence of fibrin formation is sufficient to sustain haemostasis under non-traumatic conditions.

The effect of sulfation on the anticoagulant and antithrombin III-binding properties of a heparin fraction with low affinity for antithrombin III

Heparin with low affinity for antithrombin III (ATIII) and devoid of anticoagulant activity was chemically oversulfated and fractionated by affinity for ATIII. The oversulfated material showed ATIII binding properties, as monitored by intrinsic fluorescence enhancement of ATIII. The fluorescence increase was comparable to that of the AT III high affinity fraction of native heparin. The estimated dissociation constants however, showed a 10-fold weaker binding of the oversulfated material to ATIII, Kd = 6.4 x 10(-8) M, as compared to native heparin, Kd = 0.63 x 10(-8) M. Concomitant with the binding-induced allosteric change in ATIII, the oversulfated material stimulated the ATIII-thrombin and ATIII-factor Xa reactions. The high affinity fractions of native heparin and the sulfated material were almost equally effective in enhancing the rate of thrombin neutralization by ATIII. However, a 3-fold faster rate of factor Xa inactivation was found with the native high affinity material.