George M. Willems

The role of intrinsic binding rate and transport rate in the adsorption of prothrombin, albumin, and fibrinogen to phospholipid bilayers

Abstract The rates of adsorption and desorption of prothrombin, albumin, and fibrinogen are studied by means of ellipsometry. The adsorbing surface consists of a chromium slide covered with a bilayer of negatively charged phospholipid (dioleoylphosphatidylserine) in a stirred buffer solution. Using an unstirred layer model, a simple graphical representation of the results is presented which allows detection of a transport limitation in sorption kinetics and also allows direct estimation of the thickness δ of the unstirred layer. Values of δ ranged from 2.5 to 10 μm, depending on the rate of stirring. The initial rate of prothrombin adsorption is transport-limited under all conditions studied. Adsorption of fibrinogen is much slower and determined by the intrinsic rate of protein binding. However, the adsorption becomes faster after lowering the pH and ionic strength of the buffer solution and eventually becomes transport-limited. Adsorption of albumin is slow and determined by the intrinsic binding rate for all conditions studied. Reversible adsorption is demonstrated for prothrombin, while the adsorption of fibrinogen and albumin was apparently irreversible.

Complexes of anti‐prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces

We investigated the mechanism by which anti‐prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity‐purified anti‐prothrombin antibodies from LAC‐positive plasma samples (α‐FII‐LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified α‐FII‐LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when α‐FII‐LAC+ were added to prothrombin‐deficient plasma or when purified anti‐prothrombin antibodies from LAC‐negative plasma samples (α‐FII‐LAC−) were added. When a combination of prothrombin and α‐FII‐LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The α‐FII‐LAC+ alone or a combination of prothrombin and α‐FII‐LAC− did not show inhibition. Ellipsometry studies showed that, in the presence of α‐FII‐LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with α‐FII‐LAC−. Our results show that complexes of prothrombin and anti‐prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti‐β2‐glycoprotein I antibodies.

Assessment of myocardial damage in patients with acute myocardial infarction by serial measurement of serum α-hydroxybutyrate dehydrogenase levels

A method is presented for routine estimation of ultimate infarct size in patients with acute myocardial infarction (AMI). This method implies the calculation of the total quantity of alpha-hydroxybutyrate dehydrogenase (HBDH) released by the heart per liter of plasma, with the use of five to seven plasma samples per patient during the first 4 days after onset of infarction. The choice of HBDH for this purpose was motivated by a relatively small error in estimated enzyme release for slowly eliminated enzymes. The practicality of this method was studied in the coronary care unit at Leiden University Hospital where, in 1979, 146 patients with AMI were included in the study. In 100 patients (68%) HBDH-infarct size could be calculated precisely, and in 12 other patients (8%) the assessment of HBDH-infarct size was less accurate. In 34 patients (23%), HBDH-infarct size was unobtainable because of early death (eight cases), infarctions too small in size to assess or nonexistent (six cases), too much time elapsed since infarction (six cases), and incomplete plasma sampling (14 cases). Analysis of data shows that the larger HBDH-infarct sizes were found to be associated significantly with signs of heart failure, low stroke index and low ejection fraction, presence of tachycardias and interventricular conduction disturbances, high score of left ventricular wall motion abnormalities, large myocardial perfusion defect measured with thallium-201, in-hospital death, and death in the first year after infarction. A low but significant correlation was observed between HBDH-infarct size and the severity of coronary arterial lesions.

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.

Ragged spiking of free calcium in ADP‐stimulated human platelets: regulation of puff‐like calcium signals in vitro and ex vivo

1 Human platelets respond to agonists of G protein (Gq)‐coupled receptors by generating an irregular pattern of spiking changes in cytosolic Ca2+ ([Ca2+]i). We have investigated the ADP‐induced Ca2+ responses of single, Fluo‐3‐loaded platelets in the presence or absence of autologous plasma or whole blood under flow conditions. 2 In plasma‐free platelets, incubated in buffer medium, baseline separated [Ca2+]i peaks always consisted of a rapid rising phase (median time 0.8 s) which was abruptly followed by a slower, mono‐exponential decay phase. The decay constant differed from platelet to platelet, ranging from 0.23 ± 0.02 to 0.63 ± 0.03 s−1 (mean ±s.e.m., n = 3–5), and was used to identify individual Ca2+ release events and to determine the Ca2+ fluxes of the events. 3 Confocal, high‐frequency measurements of adherent, spread platelets (diameter 3‐5 μm) indicated that different optical regions had simultaneous patterns of both low‐ and high‐amplitude Ca2+ release events. 4 With or without plasma or flowing blood, the ADP‐induced Ca2+ signals in platelets had the characteristics of irregular Ca2+ puffs as well as more regular Ca2+ oscillations. Individual [Ca2+]i peaks varied in amplitude and peak‐to‐peak interval, as observed for separated Ca2+ puffs within larger cells. On the other hand, the peaks appeared to group into periods of ragged, shorter‐interval Ca2+ release events with little integration, which were alternated with longer‐interval events. 5 We conclude that the spiking Ca2+ signal generated in these small cells has the characteristics of a ‘poor’ oscillator with an irregular frequency being reactivated from period to period. This platelet signal appears to be similar in an environment of non‐physiological buffer medium and in flowing, whole blood.

Anti‐prothrombin IgG from patients with anti‐phospholipid antibodies inhibits the inactivation of factor Va by activated protein C

Interference of anti‐phospholipid antibodies with the protein C pathway has been suggested to play a role in the development of thrombosis in the anti‐phospholipid syndrome. We studied the effect of IgG preparations containing anti‐prothrombin antibodies of 17 lupus anticoagulant‐positive patients and 12 controls on the inactivation of factor Va (FVa) by activated protein C (APC) in a system with purified coagulation factors. Test IgG was incubated with human prothrombin, phospholipid vesicles and CaCl2. Protein S, FVa and APC were added and the APC‐dependent loss of FVa activity was monitored over time. The residual amount of FVa remaining after 10 min was 14 ± 4% (mean ± SD) when 1·5 mg/ml normal IgG was present and ranged between 17% and 82% with 1·5 mg/ml patient IgG. Twelve patients IgG gave values of residual FVa >22% (i.e. 2 SD above the mean of controls), indicating that APC‐mediated inactivation of FVa was significantly inhibited. The inhibition was strictly dependent on the presence of prothrombin, proportional to the concentration of IgG and strongly diminished at a 20‐fold higher phospholipid concentration. Most, although not all, IgG containing anti‐prothrombin antibodies inhibit the APC‐catalysed FVa inactivation, which may contribute to the increased risk of thrombosis in patients with the anti‐phospholipid syndrome.

Simulation Model for Thrombin Generation in Plasma

A simulation model for the production of thrombin in plasma is presented. Values of the reaction rate constants as determined in purified systems are used and the model is tested by comparison of simulations of factor Xa, factor Va and thrombin generation curves with experimental data obtained in thromboplastin-activated plasma. Simulations of the effect of hirudin indicate that factor V is predominantly activated by thrombin and not by factor Xa. The model predicts a threshold value for the factor Xa production which, if exceeded, results in explosive and complete activation of prothrombinase. The dependence of this threshold value on different negative feedback reactions, e.g. the inactivation of thrombin and factor Xa by antithrombin III (+ heparin), is investigated. The threshold value, for control plasma in the range of 1-10 pM total factor Xa production, can be raised two orders of magnitude by accelerated inactivation of factor Xa and prothrombinase but is hardly affected by a tenfold increase in the rate of thrombin inactivation or by increased production of activated protein C. This latter effect, however, results in a more gradual input-response relation between factor Xa input and the extent of prothrombinase activation.

Enzyme tests in the evaluation of thrombolysis in acute myocardial infarction.

The activity of alpha-hydroxybutyrate dehydrogenase, creatine kinase, creatine kinase MB and aspartate aminotransferase was measured on serial plasma samples from patients with acute myocardial infarction. The study was part of a multicentre randomised trial of the effect of thrombolytic treatment in the acute phase of acute myocardial infarction. The applicability and comparability of enzyme tests for the estimation of myocardial injury were studied in 76 control patients and 74 patients treated with streptokinase. Treatment with streptokinase caused a considerable acceleration of enzyme release after acute myocardial infarction, both in patients with persistent coronary occlusion and in those with successful reperfusion. But this changed pattern of enzyme release did not affect the rate of enzyme elimination from plasma or the released proportions of different enzymes. Thus the assessment of infarct size by measurement of these enzyme activities can also be applied to patients treated with streptokinase. Moreover, the enzymes measured in the present study are all equally valid markers of myocardial injury.

Activation of blood coagulation at heparin-coated surfaces

It is hypothesized that immobilized heparin exerts a dual role in blood coagulation. On the one hand, the heparinized surface is because of its dense negative charge, thought to initiate the intrinsic pathway of blood coagulation. On the other hand, heparin is known as a potent anticoagulant drug. However, it remains to be seen how much contact-phase activation of factor XI contributes to thrombin formation and how this process is counterbalanced by which of the anti-protease activities of immobilized heparin. In the present study we examined the generation of factors XIa, IXa, and Xa, and thrombin in recalcified normal and antithrombin-depleted plasma exposed to polyacrylamide-graft polyurethane (PU) sheets modified by multipoint attachment of two different heparin species. One of them, HAH, contained the specific antithrombin binding sequence and the other one, NAH, had a low affinity for antithrombin and had no anticoagulant activity. Our data demonstrate that in contrast to PU, PU-NAH and PU-HAH are strong mediators of factor XIa and factor IXa formation in normal and antithrombin-deficient plasma. Interestingly, compared to PU-HAH and PU-NAH, thrombin formation was only slightly diminished in antithrombin-deficient plasma exposed to PU. In contrast, thrombin formation was dramatically delayed and diminished in normal plasma exposed to PU-HAH. These findings indicate that very low amounts of factor XIa apparently suffice to induce significant amounts of thrombin. In this sense, heparinized surfaces are highly thrombogenic, but our data also indicate that this activity is effectively counterbalanced by the anti-thrombin activity of the immobilized anti-coagulant species of heparin.

Complete recovery in plasma of enzymes lost from the heart after permanent coronary artery occlusion in the dog.

Plasma activities of creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBD) were measured after permanent coronary artery occlusion in the dog. Cumulative release of enzymes in plasma was calculated from these data by using a previously validated two-compartment model for circulating enzymes. Regional myocardial ischemia was measured by injection of radiolabeled microspheres. After 48 hours, the dogs were killed, and a detailed map of left ventricular enzyme activity was obtained from 108 tissue samples. Cumulative release into plasma of CK and HBD was 96 +/- 20% and 112 +/- 26%, respectively, of the total activities depleted from the heart (mean +/- SD, n = 11). The scatter in these values is inherent to the calculations, and it is concluded that both enzymes are recovered completely in plasma and, thus, can be used as quantitative markers of injury. Discrepancies between this result and earlier reports on the recovery of CK are only partly apparent and can be explained partly by underestimation of the elimination rate of CK from plasma, irregardless of tissue edema and incomplete extraction of enzyme activity from tissue.