Biomedical subjects
Publications and source records attributed to H C Hemker.
A method is described in which thrombin activity in clotting plasma can be monitored through the continuous measurement of the fluorescent split-product of the substrate Z-Gly-Gly-Arg-AMC. The signal is not impaired by turbidity; therefore proper measurement is not disturbed by the occurrence of a clot or the presence of platelets and direct measurement in platelet rich plasma is possible.
In von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 microg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases. To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; approximately 40% of vWF is required for half-normal thrombin generation in PRP. It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.
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The physiological inhibitor of tissue factor (TF).factor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPI(FL)) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TF.FVIIa-catalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TF.PC) or 25% phosphatidylserine and 75% phosphatidylcholine (TF.PSPC) was measured in the presence of preformed complexes of FXa.TFPI(FL) or FXa.TFPI(1-161) (TFPI lacking the third Kunitz domain and C terminus). At TF.PC, FXa.TFPI(FL) and FXa.TFPI(1-161) showed similar rate constants of inhibition (0.07 x 10(8) M(-1) s(-1) and 0.1 x 10(8) M(-1) s(-1), respectively). With phosphatidylserine present, the rate constant of inhibition for FXa.TFPI(FL) increased 3-fold compared with a 9-fold increase in the rate constant for FXa. TFPI(1-161). Incubation of TF.PSPC with FXa.TFPI(FL) in the absence of FVIIa followed by depletion of solution FXa.TFPI(FL) showed that FXa.TFPI(FL) remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXa.TFPI(1-161) or at TF.PC membranes. These data suggest that the membrane-bound pool of FXa.TFPI(FL) may be of physiological importance in an on-site regulation of TF.FVIIa activity.
The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K(i)(K(d)) = 14.8+/-1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M(-1)s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K(d) obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces <14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors.
Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca(2+) levels ([Ca(2+)](i)), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca(2+) two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca(2+) vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca(2+)](i) and binding of annexin V. However, a small fraction of the adherent platelets ( approximately 1%) responded much faster. Ionomycin-enhanced influx of Ca(2+) in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0+/-0.7 microm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca(2+)](i) and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca(2+)-induced generation of procoagulant sites and Ca(2+)-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall.
Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by approximately 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + alphavbeta3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (vWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from alphavbeta3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated vWF levels.
Low-molecular-weight heparins (LMWHs), like unfractionated heparin (UFH), exert their action primarily by accelerating the interaction between antithrombin (AT) and thrombin. At the levels of aXa activity that are attained in human pharmacology, it does not cause significant (>15%) inhibition of the clotting system. The essential differences between LMWHs and UFH are: (a) LMWHs attain higher plasma concentrations after subcutaneous injection (high bioavailability), and (b) in contrast to LMWHs, UFH contains very large heparin molecules with a putative hemorrhagic action. The reputedly higher aXa activity of LMWH can be shown to be largely due to the absence of Ca(2+) using the current laboratory methods to estimate this activity. Via this artifact the apparently high aXa activity of LMWHs is correlated but not related to their favorable pharmacokinetic properties. Consequently dosage guidelines for the use of different LMWHs cannot be based upon their aXa activity. Until better laboratory methods are available, clinical results are the only reliable guideline to heparin dosage.
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Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor VLeiden). Individuals without factor VLeiden who exhibit a response in functional APC-resistance tests similar to that of carriers of factor VLeiden are considered to be acquired APC resistant. This phenomenon is particularly observed in women using oral contraceptives (OC). In the present study we compared the response to APC in plasma from normal individuals, carriers of factor VLeiden and women who use OC using functional tests that either quantify the effect of APC on the endogenous thrombin potential (ETP) or on the activated partial thromboplastin time (aPTT). Both tests discriminated equally well between individuals with and without factor VLeiden who were not using OC. In contrast to the aPTT-based test, the ETP-based assay yielded significant differences in sensitivity to APC between non-OC users and OC users and between users of second and third generation OC. Since there was no correlation between APC-sensitivity determined with both assays in non-carriers of factor VLeiden and in women who use OC and a poor correlation in carriers of factor VLeiden, we propose that other plasma components differentially modulate the response to APC in the aPTT- and ETP-based APC-resistance tests and that OC change the level of plasma protein(s) that modulate the effect of APC on thrombin formation initiated via the extrinsic coagulation pathway.
Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.
Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5-80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1alpha and TNF-alpha were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from gamma-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and gamma-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.
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.
During factor Xa-catalyzed prothrombin activation, several other reaction products accumulate as a result of proteolysis of prothrombin and its activation products by thrombin and meizothrombin. Gel electrophoretic analysis and N-terminal sequencing of reaction products showed that in the absence of Ca2+ ions thrombin cleaved the following peptide bonds: Arg51-Thr52/Arg54-Asp55 in the fragment 1 (F1) domain (k = 0.4 x 10(4) M-1 s-1), Arg155-Ser156 in prothrombin (k = 2 x 10(4) M-1 s-1), and Arg284-Thr285 in prethrombin 1 (k = 0.02 x 10(4) M-1 s-1). In the presence of 2.5 mM CaCl2, cleavage in fragment 1 (Arg51-Thr52/Arg54-Asp55) was not detectable, whereas cleavage at Arg155-Ser156 (i.e., removal of F1) was inhibited 25-fold. Cleavage at Arg284-Thr285 (formation of prethrombin 2 des-1-13) was not affected by the presence of Ca2+ ions. Meizothrombin rapidly converted itself into meizothrombin des-F1. The half-life (t1/2 = approximately 30 s) of this reaction was independent of the meizothrombin concentration (0.1-1 microM meizothrombin), which is indicative for intramolecular autocatalysis (k = 0.02 s-1 in the presence of 2.5 mM Ca2+ ions). Since the rapid removal of fragment 1 precludes investigations of the cleavage at Arg284-Thr285 in intact meizothrombin, we analyzed the cleavage of this peptide bond in R155A-meizothrombin, a recombinant product that is resistant to autocatalytic removal of the fragment 1 domain. In the absence of phospholipids, R155A-meizothrombin converted itself into thrombin des-1-13 by a combination of intramolecular (k = 0.8 x 10(-4) s-1) and intermolecular autocatalysis (k = 0.2 x 10(3) M-1 s-1). Intramolecular autocatalytic conversion of R155A-meizothrombin into thrombin was not affected by the presence of phospholipids (k = 0.8 x 10(-4) s-1), whereas intermolecular autocatalysis was accelerated 25-fold (k = 5.6 x 10(3) M-1 s-1) by phospholipid vesicles. Since factor Xa/Va-catalyzed conversion of meizothrombin into thrombin occurs with k = 5.5 x 10(8) M-1 s-1, we conclude that in reaction systems containing purified proteins autocatalysis of meizothrombin hardly contributes to thrombin formation during factor Xa-catalyzed prothrombin activation.
APC resistance is often associated with the occurrence of a single point mutation in factor V (factor VLeiden) at a predominant cleavage site for the natural anticoagulant, activated protein C (APC). In this article we will discuss the effects of this mutation (Arg506-->Gln) on the down-regulation of factor Va cofactor activity and on thrombin formation by APC in model systems and in plasma. Our studies on the effects of APC on thrombin formation in plasma resulted in the development of a new method for the screening of APC resistance that is based on measurement of the effect of APC on the endogenous thrombin potential (the time integral of thrombin generated in clotting plasma). It appeared that sensitivities for APC determined via this method were considerably affected by the use of oral contraceptives (OC) and that women who use OC become acquired APC resistant. The fact that acquired APC resistance in women who use third-generation OC was more pronounced than in users of second-generation OC may explain the further increased risk for venous thrombosis associated with the use of third-generation OC.
Tissue factor:factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa:TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa:TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa:TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Argl99-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa:TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa:TFPI towards TF:factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF:factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.
Infusion of the GPIIb/IIIa-inhibitor MK383 inhibits thrombin generation in platelet rich plasma by interfering with the production of platelet procoagulant phospholipid exposure. The effect is similar to that of 0.2 U/ml of heparin. Heparin infusion, well known to inhibit thrombin generation by fostering antithrombin activity, inhibits the formation of platelet-derived procoagulant microparticles, probably by decreasing the formation of free thrombin, which, under our circumstances, is the main platelet activator.
The venom of Echis multisquamatus (Central Asian sand viper) contains a single prothrombin activator, designated multisquamase, which is structurally and functionally different from ecarin, the prothrombin activator from the venom of Echis carinatus (saw-scaled viper). Multisquamase is comprised of a 58000 Mr and a 23000 Mr subunit that consists of two disulfide-linked chains of 12000 Mr and 10000 Mr, respectively. In contrast to ecarin, which activates prothrombin and prethrombin 1 at comparable rates, and whose activity is hardly affected by Ca2+ or by changes in ionic strength, multisquamase hardly activates prethrombin 1; prothrombin activation requires Ca2+ and is strongly inhibited at high ionic strength. The most favourable kinetic parameters are observed at 1 mM Ca2+ and at low ionic strength (Km=0.085 microM and kcat=0.68 s(-1) at I approximately 0.04). An increase in ionic strength considerably reduces the rate of prothrombin activation, due to an increase of the Km (Km=0.8 microM and kcat=1.03 s(-1) at I approximately 0.2). Studies in plasmas from patients on oral anticoagulant therapy show that E. Multisquamatus venom only activates carboxylated prothrombin, whereas E. carinatus activates both prothrombin and descarboxyprothrombin. Thus, multisquamase-dependent prothrombin activation appears to require post-translational modification of the gla-domain. This venom prothrombin activator may, therefore, become a useful tool to quantitate prothrombin and descarboxyprothrombin in cases where vitamin K-dependent carboxylation of prothrombin is impaired.