Biomedical subjects
Publications and source records attributed to P G de Groot.
An ideal antithrombotic drug should inhibit thrombosis without affecting hemostasis. It should have a long half life. It should be absorbed after oral administration; it should be safe and it should have a wide therapeutic range. These criteria are discussed using new antithrombotics as examples.
We have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen- and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s-1 and 1,300 s-1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s-1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAKQAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb:IIIa, as the primary receptor responsible for platelet:fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.
The effect of sera and IgG from 12 patients with systemic lupus erythematosus (SLE) on the endothelial cell (EC) procoagulant activity (PCA) was investigated in an in vitro thrombosis model. Six of the 12 SLE sera contained antiphospholipid antibodies (aPL). EC were stimulated for 8 h at 37 degrees C with or without 50 pM tumor necrosis factor (TNF) in culture medium containing 20% patient or control serum. Then the endothelial cell matrix (ECM) was isolated and subsequently exposed in a perfusion chamber to circulating normal whole blood, anticoagulated with low molecular weight heparin (LMWH). The PCA of the ECM was determined as the amount of generated fibrinopeptide A (FPA) in samples taken before and after perfusion. Furthermore, cross sections were made of the perfused matrix and analyzed for platelet adhesion and aggregate formation. All six aPL containing sera induced a small, but significant increase of ECM procoagulant activity. When added in combination with a low dose of TNF (50 pM), a synergistic enhancement of ECM procoagulant activity was found. The FPA generation was increased to 150-614% from the values obtained after stimulation with TNF and control serum. Also a shift towards the formation of larger platelet thrombi was observed. After stimulation with TNF and patient serum the surface of ECM covered with large aggregates (greater than 5 microns) was increased by 124-329% compared to the results obtained after stimulation with control serum and TNF. When patient sera were depleted from IgG the effects were strongly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
Platelet adhesion to the vessel wall is initiated by transport of blood platelets from the bulk flow to the wall. The process of diffusion and convection of the platelets is affected by rheological conditions such as well shear rate, red blood cell (RBC) deformability, and viscosity of the medium. To study the effect of plasma viscosity on platelet adhesion, perfusion experiments with a rectangular perfusion chamber were performed. Reconstituted blood, consisting of washed platelets and washed RBCs, was circulated through this chamber for 5 minutes at a wall shear rate of 300 s-1. Different albumin concentrations were made, to obtain different medium viscosities (0.89 to 1.85 mPa.s). Platelet adhesion decreased with increasing medium viscosity up to viscosities of 0.95 mPa.s, but increased with medium viscosity above this value. Instead of human albumin solution, different plasma viscosities were obtained by dilution of Waldenström plasma with buffer. Plasma was depleted of fibronectin, which gave a final plasma viscosity of 2.0 mPa.s, and was dialyzed against HEPES buffer and subsequently diluted with the dialysis buffer in different fractions (0.89 to 2.00 mPa.s). Perfusions were performed over a purified von Willebrand factor coating on glass, or over an endothelial cell matrix, preincubated with von Willebrand factor. With both surfaces, platelet adhesion was dependent on the plasma viscosity in a similar way: at low plasma viscosities, adhesion was decreased with increasing plasma viscosity, while at higher plasma viscosities, adhesion increased with plasma viscosity. Adhesion values at higher plasma viscosity or at higher human albumin concentrations could be explained by effects of the medium on the rigidity of the RBCs, since platelet adhesion is known to be increased by enhanced RBC rigidity. Effects of the medium on the deformability of the RBCs were measured separately with the laser diffraction method. These experiments confirmed that presence of human albumin or plasma in the measuring suspension increased the rigidity of RBCs. To prevent influence of the medium on the RBCs in perfusion experiments, the RBCs were fixated with glutaraldehyde. Perfusion experiments with fixated RBCs in plasma over a von Willebrand factor preincubated endothelial cell matrix, showed a consequent decrease in adhesion with increasing plasma viscosity, according to the diffusion theories, whereas the increase of adhesion at high plasma viscosities was lacking. This suggests that the latter effect was entirely due to increased transport of platelets by more rigid RBCs.
Antiphospholipid antibodies (aPL) are defined by anticardiolipin antibody (aCL) ELISA and prolongation of phospholipid dependent coagulation assays (lupus anticoagulant; LAC). For the binding of aCL to cardiolipin a cofactor, beta 2-glycoprotein I (beta 2-GPI), is necessary. We have investigated whether the same cofactor is essential for LAC activity. Plasma from 6 LAC positive patients and 3 controls was depleted from beta 2-GPI by means of affinity chromatography. From the 6 LAC positive plasmas, 4 became LAC negative (tested with dRVVT) when beta 2-GPI was depleted and became positive again when purified beta 2-GPI (200 micrograms/ml) was added. A dose response curve showed that addition of 50 micrograms/ml beta 2-GPI to beta 2-GPI deficient patient plasma, led to a positive dRVVT. Depletion of, and addition of beta 2-GPI to plasma from controls had no effect on the dRVVT. Measurement of beta 2-GPI plasma levels in 19 LAC positive patients, 40 LAC negative patients and 15 controls showed no difference in beta 2-GPI levels. These results show that a combination of aPL and beta 2-GPI is essential not only for binding to cardiolipin, but also for LAC activity and imply that low beta 2-GPI levels (less than 50 micrograms/ml) can lead to false negative LAC tests. These observations may lead to new insights in the pathophysiological complications associated with aPL.
The adhesion of platelets to purified laminin under flow conditions was investigated. Adhesion to laminin was strongly dependent on the presence of divalent cations. In the absence of cations platelet adhesion (8% coverage in 5 minutes) was maximal at a shear rate of 100/s and no adhesion could be detected at shear rates above 800/s. In the presence of 0.8 mmol/L Mg2+ and 2 mmol/L Ca2+ platelet adhesion reached its maximum (30% coverage) around 800/s. At 1,800/s platelets still adhered to purified laminin (coverage of 6%). Antibodies against the E8 domain of laminin and antibodies against the alpha 6 and beta 1 chains of platelet membrane glycoprotein very late activation antigen-6 (VLA-6), completely inhibited adhesion. No inhibition was found with antibodies against glycoprotein IIb:IIIa, against the alpha 2 chain of VLA-2, and against the alpha 5 chain of VLA-5. Fibronectin and von Willebrand factor were not involved in laminin-dependent adhesion. Anti-VLA-6 partly inhibited platelet adhesion to the extracellular matrix of endothelial cells at shear rates below 800/s. Preincubation of the matrices with antilaminin E8 antibodies did not influence the adhesion. These results show that purified laminin supports platelet adhesion and that the presence of VLA-6 is important for platelet adhesion under flow conditions. The protein in the matrix with which VLA-6 interacts is currently unknown.
The interaction of vitronectin-thrombin-antithrombin III (VN.TAT) complex with endothelial cells (EC) was investigated. Binding was specific and time- and concentration-dependent. Kinetics revealed an apparent dissociation constant of 16 nM and 1.7 x 10(5) binding sites/endothelial cell. The binding determinant of the ternary complex was located on the VN moiety. Since the association of VN to TAT adds its specific properties to the VN.TAT complex, the involvement of the heparin binding domain and the cell attachment site of VN was investigated. Neither addition of RGD peptide nor blocking of the vitronectin receptor with a monoclonal antibody interfered with VN.TAT binding to EC. Addition of heparin, a VN-derived peptide comprising two heparin binding consensus sequences or a monoclonal antibody directed against the heparin binding domain on VN, completely inhibited VN.TAT binding to EC. These results indicate that the interaction is mediated through the heparin binding domain of VN. Digestion of heparan sulfate proteoglycans resulted in a decrease of VN.TAT binding to EC, indicating the involvement of heparin-like structures on the EC surface. Our findings point to an unrecognized mechanism by which VN may act as scavenger in order to enhance the clearance of end products of the clotting system via binding of the ternary VN.TAT complex to the luminal surface of EC.
Von Willebrand's disease (vWD) 'Vicenza' is characterized by low plasma von Willebrand Factor antigen (vWF:Ag) and very low levels of Ristocetin Cofactor activity (RiCof). The hemorrhagic tendency in vWD 'Vicenza' is, however, mild and bleeding times in this rare vWD-subtype are only slightly prolonged (1). Larger than normal multimers of plasma-vWF and normal levels of platelet-vWF have both been suggested to compensate the defects that are normally present in vWD. To elucidate the mechanisms involved, whole blood of four patients with vWD 'Vicenza' was circulated through a rectangular perfusion chamber (2) with fibrillar collagen as adhesive surface. Under these conditions, both plasma and platelet vWF participate to platelet adhesion. Compared to perfusion results with blood of normal donors, platelet adhesion of 'Vicenza' patients was decreased. However, the Vicenza defect was less than was observed in parallel experiments with blood of vWD type 1 subtype platelet-low patients and blood of a severe vWD patient. Adhesion was not increased in perfusions with only plasma of the 'Vicenza' vWD-patients. Equal vWF:Ag concentrations of normal multimeric composition were just as effective as the high multimeric 'Vicenza' vWF. Therefore, the abnormal plasma-vWF in 'Vicenza' patients does not cause the relatively high adhesion obtained with whole blood. In contrast, platelets of vWD 'Vicenza' patients resuspended in human albumine solution (HAS) showed far better adhesion than (vWF-poor) platelets of a patient with severe vWD. The values were at least comparable and tended to be higher than those obtained with normal platelets or with platelets of patients with vWD type I subtype platelet normal.(ABSTRACT TRUNCATED AT 250 WORDS)
OBJECTIVES: A prospective, controlled multicenter study was performed to estimate the obstetric risks of antiphospholipid antibodies (the lupus anticoagulant and anticardiolipin antibodies). In addition, the risks of prior thrombosis, obstetric history, systemic lupus erythematosus, and high-dose prednisone treatment were evaluated. STUDY DESIGN: After screening for antiphospholipid antibodies in patients with lupus erythematosus or women with prior fetal loss(es), 59 subsequent pregnancies with and 54 without these antibodies were followed. RESULTS: The presence of the lupus anticoagulant and a history of at least three spontaneous abortions could predict fetal loss (p = 0.032 and 0.001, respectively). In live born infants, a low birth weight could be predicted by the presence of anticardiolipin antibodies (p = 0.034), prior intrauterine fetal death (p = 0.025), and treatment with high-dose prednisone (p = 0.002). No relationships were seen between antiphospholipid antibodies and small-for-gestational-age newborns and pregnancy-induced hypertension or preeclampsia. The disappearance of antiphospholipid antibodies during pregnancy was not correlated with live birth. CONCLUSION: It is concluded that the presence of antiphospholipid antibodies is a risk factor for adverse pregnancy outcome.
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Fluctuations of lupus anticoagulant activity and anticardiolipin antibody titres were studied in 53 patients with systemic lupus erythematosus (SLE). The median study time was 26 months with a median number of 12 samples. Lupus anticoagulant was measured by the kaolin clotting time (KCT) and dilute Russell viper venom time (dRVVT) assays; anticardiolipin antibodies were assayed by an enzyme linked immunosorbent assay (ELISA). Normal and increased KCTs or dRVVTs were seen during follow up in 13 and 12 patients, respectively. IgG anticardiolipin antibodies changed from negative to positive or positive to negative in 26 patients and IgM anticardiolipin antibodies in 16 patients. Disease activity and treatment with prednisone could account for these fluctuations in the kaolin clotting time (KCT) in 7 of 13 patients and in the dRVVT in 2 of 12 patients. Whole group analysis showed that the KCT, dRVVT, and IgM anticardiolipin antibodies were not associated with disease activity, in contrast with IgG anticardiolipin antibodies. During treatment with prednisone normal KCT and dRVVT results were obtained more easily than normal anticardiolipin antibody levels. It is recommended that lupus patients should not be classified as antiphospholipid antibody positive or negative on the basis of only one sample.
Cell seeding may decrease the thrombogenicity of implanted vascular grafts, but its application is hampered by the limited availability of autologous endothelial cells. We studied the interaction of alternate cells, human peritoneal mesothelial cells, with whole blood in a flow chamber. When citrated blood was perfused over mesothelial cells, platelet adhesion was seen on the intercellular matrix but not on the cells themselves. Perfusions with blood anticoagulated with low-molecular-weight heparin resulted in fibrin formation at the surface of mesothelial cells but not at the surface of human umbilical venous endothelial cells. At shear rates of 200 sec-1 fibrin deposition on the mesothelial cell surface increased during the first 5 minutes to 5.7 +/- 1.06 micrograms fibrin per square centimeter, whereafter these values stabilized. The procoagulant activity of cultured mesothelial cells was higher than that of peritoneal membrane studied ex vivo. However, cultured mesothelial cells incubated with polyclonal antibodies against tissue factor showed a significant decrease in procoagulant activity. We conclude that human peritoneal mesothelial cells may be used for cell seeding procedures, provided that their tissue factor expression can be controlled.
BACKGROUND: Nitric oxide (NO) has been identified as endothelium-derived relaxing factor (EDRF), which, in addition to its relaxant effects on vascular smooth muscle cells, is also a potent inhibitor of platelet aggregation. An inhibitory role on platelet adhesion has been suggested from experiments with washed platelets under static conditions. We have determined whether endothelium-derived and exogenous NO also regulates platelet adhesion in whole blood under flow conditions. METHODS AND RESULTS: The effect of endothelium-derived NO was studied by the addition of specific inhibitors of NO production, L-N-monomethyl arginine (L-NMMA) and N-iminoethyl-L-ornithine (L-NIO), to a perfusion system in which both endothelial cells and their matrices were present. A concentration-dependent increase in platelet adhesion to the matrix was found with a maximum inhibition at a concentration of 2 mM L-NMMA and 0.1 mM L-NIO. The effect was dependent on the presence of endothelial cells, because no increase in platelet adhesion was observed in their absence. The effect of exogenous NO was tested in a specially devised perfusion system in which the NO was introduced at the site of adhesion by means of a porous membrane on which an extracellular matrix of endothelial cells was present. Inhibition of platelet adhesion by NO was found at all shear rates tested and after all perfusion periods. CONCLUSIONS: These results demonstrate that NO is a potent inhibitor of platelet adhesion under flow conditions and thereby contributes to the regulatory role of vascular endothelial cells on platelet-vessel wall interaction.
In the present study, uremic patients on chronic maintenance hemodialysis were treated with recombinant erythropoietin. Before and after 20 weeks of treatment, platelet adhesion and aggregation were studied with perfusions over a sprayed collagen surface and over matrix of cultured endothelial cells with high tissue factor activity. The influence of the erythropoietin induced raise in hematocrit on platelet transport and adhesion was excluded by performing the perfusions at a standard red blood cell concentration. The present study clearly demonstrates that erythropoietin treatment improves platelet adhesion and aggregation in addition to and independent of its effect on the hematocrit. Studies with control platelets resuspended in plasma of untreated patients showed that a uremic plasma factor reduced adhesion and thrombin- and collagen-dependent aggregation. Patient platelets resuspended in control plasma showed no defects. After erythropoietin treatment, the plasma-induced inhibition of adhesion and aggregation had almost completely disappeared from patient plasma. The beneficial effect of the erythropoietin treatment on uremic hemostasis is therefore twofold. The increase of the red blood cell mass improves transport of platelets, and thus adhesion to the vessel wall. The intrinsic defect due to the presence of an inhibitory toxin in uremic plasma is, in large part, corrected. Improved neutralization of uremic toxins by red blood cells or less production of toxins by better oxygenated tissue might play a role in the observed phenomena.
The effect of sera and purified IgG isolated from plasma of 46 patients with systemic lupus erythematosus (SLE) and 9 healthy donors on the endothelial cell (EC) mediated protein C activation was investigated. Out of the 46 SLE sera used, 19 were antiphospholipid antibodies (aPL) positive. From 12 patients IgG was isolated, of which 6 contained aPL. EC were first incubated with IgG (7 mg/ml) or serum (1:1 diluted) for 1 h and then tested for their ability to promote protein C activation by thrombin, with the cells either in a monolayer or in a suspension. The normal range (mean of control values +/- 2 SD) of protein C activation was 80-120%. In contrast to others, we could not detect an inhibition of protein C activation by any of the patient IgG's or sera. The recently described cofactor for binding of antiphospholipid antibodies to phospholipids, beta 2-glycoprotein I, was purified and added to the purified IgG's. A combination of these two components did not inhibit the EC mediated protein C activation by thrombin. This study suggests that the inhibition of the protein C activation, mediated by EC, is not a general mechanism by which aPL related thrombosis can be explained.
We have studied the influence of ascorbate on extracellular matrix formation in cultured human endothelial cells, smooth muscle cells and fibroblasts and measured the influence of the changed composition of their isolated extracellular matrices on their affinity for platelets. When endothelial cells were grown for a week in the presence of ascorbate, no influence on proline incorporation in their extracellular matrix was found. In accordance, no influence on platelet adhesion or aggregate formation on these matrices was detected. When smooth muscle cells were cultured in the presence of ascorbate, a strong increase in the amount of collagen types I and III in the extracellular matrix was found. When these matrices were perfused with whole blood, a significant enhanced increase in aggregate formation was observed. No influence was seen on the total coverage of the matrix with platelets. When fibroblasts were grown in the presence of ascorbate, no significant increase in proline incorporation in their matrix was measured. However, an increased adhesion of platelets was seen to the matrices at lower shear rates. We conclude that ascorbate feeding has a significant effect on endogenous deposited matrices of smooth muscle cells and fibroblasts, and that the changed composition had profound effects on platelet interaction with these matrices.
Infusion of tumor necrosis factor (TNF) into tumor-bearing mice led to intravascular clot formation with fibrin deposition in microvessels in the tumor bed in close association with the vessel wall, which could be prevented by active site-blocked factor IXa (IXai). This observation prompted us to examine the role of the intrinsic system in activation of the coagulation mechanism on TNF-stimulated human endothelial cell monolayers and endothelial-derived matrix during exposure to purified coagulation factors or flowing blood. Treatment of endothelial cells in intact monolayers with TNF induced expression of the procoagulant cofactor tissue factor (TF) in a dose-dependent manner, and after removal of the cells, TF was present in the matrix. TNF-treated endothelial cell monolayers exposed to blood anticoagulated with low molecular weight heparin induced activation of coagulation. Addition of IXai blocked the procoagulant response on TNF-treated endothelial cells, and consistent with this, the presence of factor IX/VIIIa enhanced endothelial TF/factor VII(a) factor X activation over a wide range of cytokine concentrations (0-600 pM). When TF-dependent factor X activation on endothelial cells was compared with preparations of subendothelium, the extracellular matrix was 10-20 times more effective. IXai blocked TF/factor VII(a) mediated activated coagulation on matrix, but only at lower concentration of TNF (less than 50 pM). Similarly, enhancement of factor Xa formation on matrix by factors IX/VIIIa was most evident at lower TNF concentrations. When anticoagulated whole blood flowing with a shear of 300 s-1 was exposed to matrices from TNF-treated endothelial cells, but not matrices from control cells, fibrinopeptide A (FPA) generation, fibrin deposition, and platelet aggregate formation were observed. FPA generation could be prevented by a blocking antibody to TF and by active site-blocked factor Xa (Xai) over a wide range of TNF concentrations (0-600 pM), whereas IXai only blocked FPA generation at lower TNF concentrations (less than 50 pM). Activation of coagulation on matrix from TNF-stimulated endothelial cells was dependent on the presence of platelets, indicating the important role of platelets in propagating the reactions leading to fibrin formation. These observations demonstrate the potential of cytokine-stimulated endothelium and their matrix to activate coagulation and suggest the importance of the intrinsic system in factor Xa formation on cellular surfaces.
Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme-linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.