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P G de Groot

Publications and source records attributed to P G de Groot.

At least 19 recordsLinked to original sources
PubMed1996

Platelet adhesion to collagen type IV under flow conditions.

Collagen type IV is a sheet-forming collagen and a major constituent of the vessel wall. To find out which conditions are important for platelet adhesion to collagen type IV, we performed perfusion studies with anticoagulated blood in parallel plate perfusion chambers. The role of divalent cations was investigated by using plasmas with variable concentrations of Mg2+ and Ca2+ ions. When Mg2+ concentration was decreased from 2.00 mmol/L to 0.25 mmol/L at a fixed Ca2+ concentration of 1.25 mmol/L, platelet coverage on the collagen type IV surface decreased from 22.8% +/- 1.8% (n = 4) to 4.6% +/- 0.6% (n = 4) at a shear rate of 1,600 s-1. Also, platelet aggregate formation on collagen type IV was strongly impaired. A monoclonal antibody against the glycoprotein (Gp) Ib receptor and von Willebrand factor (vWF)-depleted plasma reduced the platelet coverage to collagen type IV to, respectively, 10% and 45% of the control value. Electron microscopy showed that vWF was only present between platelets and between the platelet and the collagen type IV surface, but did not bind elsewhere to collagen type IV. These data indicate that collagen type IV is a reactive collagen for platelets. Differences in physiologic plasma magnesium concentrations may in part explain the differences in platelet reactivity to collagen type IV between individuals, and perhaps contribute to differences in the risk for thrombosis.

Calcium↗
PubMed1996

Protein C activation on endothelial cells by prothrombin activation products generated in situ: meizothrombin is a better protein C activator than alpha-thrombin.

The conversion of protein C into activated protein C (APC) by the thrombin-thrombomodulin complex on the surface of endothelial cells initiates an essential negative feedback reaction on blood coagulation. APC, together with its non-enzymic cofactor protein S, inactivates factors Va and VIIIa, the non-enzymic protein cofactors of the prothrombinase and intrinsic tenase complex, by proteolytic degradation. In this study we report that prothrombin activation products, generated by the prothrombinase complex on the surface of quiescent endothelial cells, are able to activate protein C. Subsequent inactivation of factor Va by the APC that was formed decreased the rate of prothrombin activation, thus demonstrating in vitro the negative feedback loop on coagulation factor activation. The anticoagulant feedback reaction of APC on the prothrombinase complex was stimulated 3-4-fold by the addition of protein S but not by thrombin-cleaved protein S or by protein S complexed with C4b-binding protein. Stimulation of endothelial cells with 50 pM tumour necrosis factor (TNF) or 500 pM interleukin 1 (IL-1) resulted in a 70% decrease in activation of protein C by exogenously added alpha-thrombin, which seemed to be due to down-regulation of thrombomodulin activity on the surface of endothelial cells. However, when prothrombin activation products generated in situ were allowed to activate protein C, stimulation of endothelial cells with TNF and IL-1 resulted in only a 25% decrease in activation of protein C. Stimulation with TNF or IL-1 did not affect the ability of endothelial cells to support prothrombinase activity. We investigated whether the differences in extent of protein C activation by exogenously added alpha-thrombin and by prothrombin activation products generated in situ were due to meizothrombin formed during prothrombin activation. Previous reports from our groups revealed that meizothrombin is generated as a transient intermediate during prothrombin activation on phospholipid vesicles and endothelial cells. Here we show that meizothrombin is at least a 6-fold better activator of protein C on the surface of endothelial cells than is alpha-thrombin. These results demonstrate that meizothrombin, formed during the initial phase of prothrombin activation, efficiently down-regulates both its own formation and that of thrombin.

Cells, Cultured↗
PubMed1996

Thrombin regulation by physiological inhibitors: the role of vitronectin.

The generation of thrombin and its various activities have to be tightly controlled in the circulation as well as at extracellular sites to prevent pathological situations. Both vessel wall-associated thrombomodulin and circulating serine protease inhibitors meet the requirements for regulation of thrombin function. The final products of thrombin inhibition are ternary complexes together with the adhesion protein vitronectin. Due to a conformational switch in the vitronectin molecule, ternary complexes are endowed with heparin-binding properties and become specifically bound to cell surface sites on endothelial and other cells. This interaction appears to be responsible for the clearance and translocation of ternary complexes in the vasculature and at other sites. In addition, ternary complexes may provoke other cellular activities by binding to nonintegrin cell surface receptors. Along the vasculature, extracellular-associated vitronectin serves as a binding and stabilizing cofactor for plasminogen activator inhibitor 1, which thereby becomes a slow-reacting thrombin inhibitor. It is believed that clot-associated vitronectin-PAI-1 complex not only stabilizes initial thrombus formation, but is also responsible for thrombin neutralization and the necessary switch towards fibrinolysis. Vitronectin thereby becomes an integral cofactor for regulation and control of thrombin multifunctional activities.

Animals↗
PubMed1996

Adhesion of blood platelets is inhibited by VCL, a recombinant fragment (leucine504 to lysine728) of von Willebrand factor.

VCL, fragment Leu504 to Lys728 of von Willebrand factor (vWF) expressed in Escherichia coli, contains the glycoprotein (GP) Ib-binding domain of vWF. This fragment inhibited ristocetin-induced platelet aggregation with an IC50 of 0.2 mumol/L and botrocetin-induced platelet aggregation with an IC50 of 0.08 mumol/L. We studied the antiadhesive profile of VCL by adding it to blood that was circulated over various adhesive surfaces. VCL inhibited adhesion to endothelial cell matrix, which served as a model of the vessel wall. Maximal inhibition at a high shear rate of 1600 s-1 was stronger (60%) than at a low shear rate of 300 s-1 (40%). Half maximal inhibition was found to be 1.5 mumol/L at both shear rates. The role of various adhesive molecules was investigated in more detail by coating glass coverslips with collagen type I, laminin, fibronectin, or vWF. Fibrinogen was studied as well. Platelet adhesion to laminin and vWF was not inhibited by VCL. Adhesion to collagen, fibronectin, and fibrinogen was particularly inhibited at a high shear rate. VCL coated to a coverslip caused a concentration-dependent adhesion that was blocked by antibodies against GPIb, which block interaction with vWF. Binding studies showed a nonsaturable ristocetin binding of VCL to platelets that was blocked by vWF or inhibitory antibodies against GPIb. Binding to collagen was weak, and VCL did not inhibit binding of vWF at a 5000-fold excess. From these data, we conclude that VCL inhibits adhesion in all cases in which adhesion is vWF dependent by competing for vWF binding to activated GPIb. The lack of inhibition of adhesion to vWF as a single molecule may be explained by assuming that this adhesion is determined by interaction of nonactivated GPIb with vWF that has been changed in conformation by adsorption. Studies investigating thrombus formation on the connective tissue of an atherosclerotic plaque in a human coronary artery showed that VCL was able to partially prevent this thrombus formation. VCL may be of value in preventing adhesion and thrombus formation under conditions in which these processes are dependent on vWF.

Animals↗
PubMed1996

Primary antiphospholipid syndrome evolving into systemic lupus erythematosus.

A young woman had a history of spontaneous venous thromboembolic disease which recurred on several occasions after cessation of treatment with oral anticoagulants. The presence of antiphospholipid antibodies (lupus anticoagulant and a high titre of lgG class anticardiolipin antibodies) in the absence of other clinical and serological features of systemic lupus erythematosus (SLE) confirmed a diagnosis of primary antiphospholipid syndrome (PAPS). Antinuclear antibodies (ANA) were positive (1:1280; speckled pattern). Twelve years after the first thrombotic episode she fulfilled criteria for the classification of SLE (antinuclear antibodies, platelet count < 100 x 10(9)/l, anti-dsDNA antibodies, Coombs' positive haemolytic anaemia). She suffered a myocardial infarction while adequately anticoagulated and developed polyarthritis and immune complex-mediated nephritis over the next 3 years. This case history supports suggestions made by others that a strongly positive ANA test in a patient diagnosed with PAPS may be a harbinger for the development of SLE. Such evolution can take place over more than 10 years.

Adult↗
PubMed1996

Protein C and other cofactors involved in the binding of antiphospholipid antibodies: relation to the pathogenesis of thrombosis.

In this review we will discuss the possible interference of antiphospholipid antibodies with the protein C system. Antiphospholipid antibodies can interfere with the protein C system in different ways: (i) via inhibiting the formation of thrombin; (ii) via interference with the activation of protein C by the thrombomodulin-thrombin complex; (iii) via inhibition of the assembly of the protein C complex; (iv) via inhibition of the activity of protein C, directly or via its cofactor protein S, and (v) via antibodies directed against the substrates of APC, factors Va and VIIIa, thereby protecting them for inactivation. The experimental and theoretical indications that one of these mechanisms will explain the pathogenesis of the antiphospholipid syndrome is critically examined.

Antibodies, Antiphospholipid↗
PubMed1996

Interventional thermal injury of the arterial wall: unfolding of von Willebrand factor and its increased binding to collagen after 55 degrees C heating.

PURPOSE: Thermal angioplasty alters the thrombogenicity of the arterial wall. In previous studies, platelet adhesion was found to increase after heating human subendothelium to 55 degrees C and decrease after heating to 90 degrees C. In the present electron microscopic study, the mechanism of this temperature-dependent platelet adhesion to the heated arterial wall is elucidated by investigating temperature-dependent conformational changes of von Willebrand factor (vWF) and collagen types I and III and the binding of vWF to heated collagen. METHODS: Purified vWF and/or collagen was applied to electron microscopic grids and heated by floating on a salt-solution of 37 degrees C, 55 degrees C or 90 degrees C for 15 s. After incubation with a polyclonal antibody against vWF and incubation with protein A/gold, the grids were examined by electron microscopy. RESULTS: At 37 degrees C, vWF was coiled. At 55 degrees C, vWF unfolded, whereas heating at 90 degrees C caused a reduction in antigenicity. Collagen fibers heated to 37 degrees C were 60.3 +/- 3.1 nm wide. Heating to 55 degrees C resulted in the unwinding of the fibers, increasing the width to 87.5 +/- 8.2 nm (p < 0.01). Heating to 90 degrees C resulted in denatured fibers with an enlarged width of 85.1 +/- 6.1 nm (p < 0.05). Heating of collagen to 55 degrees C resulted in an increased vWF binding as compared to collagen heated to 37 degrees C or to 90 degrees C. Incubation of collagen with vWF, prior to heating, resulted in a vWF binding after heating to 55 degrees C that was similar to the 37 degrees C binding and a decreased binding after 90 degrees C. CONCLUSIONS: After 55 degrees C heating, the von Willebrand factor molecule unfolds and collagen types I and III exhibit an increased adhesiveness for von Willebrand factor. Heating to 90 degrees C denatures von Willebrand factor and collagen. The conformation changes of von Willebrand factor and its altered binding to collagen type I and III may explain the increased and decreased platelet adhesion to subendothelium after 55 degrees C and 90 degrees C heating, respectively.

Analysis of Variance↗
PubMed1996

A3 domain is essential for interaction of von Willebrand factor with collagen type III.

von Willebrand factor (vWF) mediates platelet adhesion at sites of vascular damage. It acts as a bridge between receptors on platelets and collagens present in the connective tissue. Two collagen binding sites have been identified on the A1 and A3 domain of the vWF subunit. To study the functional importance of these binding sites, we have made two deletion mutants that lack the A1 domain (residues 478-716; delta A1-vWF; Sixma et al. Eur. J. Biochem, 196, 369, 1991 [1]) or the A3 domain (residues 910-1113; delta A3-vWF). After transfection in baby hamster kidney cells overexpressing furin, the mutants were processed and secreted efficiently. Ristocetin or botrocetin induced platelet binding was normal for delta A3-vWF as was binding to heparin and factor VIII. As reported by Sixma et al. (1) delta A1-vWF still binds to collagen type III, indicating that the A3 domain is sufficient for the interaction. In the current study, we investigated the binding of delta A3-vWF to collage type III. When preincubated on collagen type III it did not support platelet adhesion under flow conditions, whereas it was able to support platelet adhesion when coated directly to a glass surface. The binding of 125I-delta A3-vWF to collagen was specific but maximal binding was about 40 times less compared to 125I-vWF. When added at 25 times excess, delta A3-vWF did not compete with 125I-vWF for binding to collagen type III, whereas delta A1-vWF did. The binding of 125I-delta A3-vWF could be blocked by excess unlabeled vWF but not by delta A1-vWF. In conclusion, we demonstrate that the A3 domain in vWF contains the major collagen binding site. The major binding site present on the A3 domain and the minor site present on A1 bind to different sites on collagen.

Animals↗
PubMed1996

Factor V Leiden, antiphospholipid antibodies and thrombosis in systemic lupus erythematosus.

Thromboembolic complications are frequently observed in patients with systemic lupus erythematosus (SLE). Significant associations have been reported between these complications and the presence of antiphospholipid antibodies, notably the lupus anticoagulant and anticardiolipin antibodies. Factor V Leiden is a genetic disorder associated with an increased risk of venous thrombosis. We studied these factors in 173 patients with SLE in relation to both arterial and venous thrombosis. The frequency of factor V Leiden in SLE patients in comparable to that in the Dutch population (5%) and a risk factor for venous thrombosis (odds ratio 4.9; CI 1.2-19.6), but not for arterial thrombosis. The lupus anticoagulant is a risk factor for both arterial thrombosis (odds ratio 7.1: CI 2.9-17.4) and venous thrombosis (odds ratio 6.4; CI 2.7-15.4). From multivariate analysis, both the lupus anticoagulant and factor V Leiden appeared independent risk factors for venous thrombosis.

Adolescent↗
PubMed1995

Internalization of vitronectin-thrombin-antithrombin complex by endothelial cells leads to deposition of the complex into the subendothelial matrix.

Internalization of the ternary vitronectin-thrombin-antithrombin (VN-TAT) complex by human umbilical vein endothelial cells was investigated. Radiolabeled VN-TAT was bound to the cell surface at 4 degrees C, and internalization was initiated by increasing the temperature to 37 degrees C. After 30 min about half of the VN-TAT complex disappeared from the cell surface and accumulated in the subendothelial matrix. Translocation of VN-TAT complex from the luminal to the basolateral side was confirmed by electron microscopic evaluation of cross-sections of endothelial cells incubated with gold-conjugated VN-TAT complex. Furthermore, cells cultured in VN-TAT deficient serum, incubated with purified VN-TAT, and subsequently assayed for fluorescent staining using a monoclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin showed co-localization of both antibodies in punctates. Punctates were randomly distributed in both the xy and xz plane of endothelial cells as evidenced by confocal laser scanning microscopy. Trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis showed that VN-TAT was not degraded during translocation and inhibition of the microfilament system reduced release of VN-TAT to the matrix, indicating that transcytosis was responsible for translocation. These findings emphasize that VN-TAT complex is taken up by endothelial cells, not only leading to the removal of inactivated thrombin from the circulation but also to deposition of VN into the subendothelial matrix.

Antithrombin III↗
PubMed1995

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood.

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.

Bernard-Soulier Syndrome↗
PubMed1995

Platelet adhesion to fibronectin in flow: the importance of von Willebrand factor and glycoprotein Ib.

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.

Antibodies, Monoclonal↗
PubMed1995

Evidence that fibrin alpha-chain RGDX sequences are not required for platelet adhesion in flowing whole blood.

The role of the RGDX putative receptor-recognition sites, which are present on the alpha chains of fibrin, in promoting platelet adhesion has been examined in flowing whole blood using the rectangular perfusion chamber at wall shear rates of 340 and 1,600/s. Platelets adhered to a comparable extent to surfaces coated with native fibrin and surfaces coated with fragment X-fibrin, a product of limited fibrinolysis that lacks the RGDS sites normally present at positions 572 to 575 of the alpha chains. The strengths of these adhesive interactions were comparable based on the concentrations of the antiadhesive peptide D-RGDW required to block platelet deposition to native and fragment X-fibrin at both low and high wall shear rate. Blocking either or both RGDX sequences with peptide-specific monoclonal antibodies did not inhibit platelet deposition in perfusion experiments performed with normal blood at 340/s, indicating that neither RGD motif is required for adhesion. However, adhesion was partly inhibited by anti-RGDX antibodies when perfusions were performed with blood from an afibrinogenemic patient, suggesting the RGDX sequences may play a limited role in platelet deposition. Exposure of fibrin surfaces to plasminogen/tissue-type plasminogen activator did cause a time-dependent loss of adhesiveness, but this effect was only weakly correlated with proteolysis of the fibrin alpha chains. These observations provide evidence that neither RGDX sequence is required for platelets to adhere avidly to fibrin in flowing blood. These results further suggest that incomplete fibrinolysis yields a highly thrombogenic surface.

Amino Acid Sequence↗
PubMed1995

Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor.

To assess the relative importance of the glycoprotein (GP) Ib binding domain and the RGDS binding site in platelet adhesion to isolated von Willebrand factor (vWF) and to collagen preincubated with vWF, we deleted the A1 domain yielding delta A1-vWF and introduced an aspartate-to-glycine substitution in the RGDS sequence by site-directed mutagenesis (RGGS-vWF). Recombinant delta A1-vWF and RGGS-vWF, purified from transfected baby hamster kidney cells, were compared with recombinant wild-type vWF (WT-vWF) in platelet adhesion under static and flow conditions. Purified mutants were coated on glass or on a collagen type III surface and exposed to circulating blood in a perfusion system. Platelet adhesion under static condition, under flow conditions, and in vWF-dependent adhesion to collagen has an absolute requirement for GPIb-vWF interaction. The GPIIb/IIIa-vWF interaction is required for adhesion to coated vWF under flow conditions. Under static condition and vWF-dependent adhesion to collagen, platelet adhesion to RGGS-vWF is similar as to WT-vWF, but platelet spreading and aggregation are abolished.

Amino Acid Sequence↗
PubMed1995

Cultured endothelial cells regulate platelet adhesion to their extracellular matrix by regulating its von Willebrand factor content.

Endothelial cells and their extracellular matrix formed in vitro are often used as a model for subendothelium in studies on platelet-vessel wall interaction. We have characterized the influence of culture conditions of endothelial cells on the formation of extracellular matrix and on the interaction of the matrix with platelets. Passage number, time of confluence, serum concentration and the addition of heparin, growth factors and antibiotics to the culture medium were varied and the extracellular matrices were isolated. The amount of fibronectin and von Willebrand factor present in the matrix were measured and the number of platelets adhering to these matrices after perfusion with citrated whole blood at a shear rate of 1000 s-1 was determined. A three times increase of the amount of von Willebrand factor in the matrix was found when the serum concentration was increased from 2.5% to 30%. When the passage number of the cells was increased or the period during which the cells were at confluence was extended, the amount of von Willebrand factor in the matrix was decreased up to 50%. Addition of heparin or ECGS (endothelial cell growth supplement) decreased the von Willebrand factor content in the matrix. Addition of penicillin or streptomycin to the culture medium had no influence on the amount of von Willebrand factor deposited in the matrix or secreted into the medium, however, other antibiotics such as gentamycin and neomycin decrease the amount of von Willebrand factor in the matrix. No influence on the amount of fibronectin in the matrix was found under all conditions tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1995

Platelet adhesion.

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Endothelium, Vascular↗
PubMed1995

Tissue factor expression on mesothelial cells is induced during in vitro culture--manipulation of culture conditions creates perspectives for mesothelial cells as a source for cell seeding procedures on vascular grafts.

Lining the luminal surface of prosthetic small diameter bypasses with endothelial cells (EC) will lower its thrombogenicity. Unfortunately, human EC are only scarcely available. Mesothelial cells (MC) have antithrombotic properties in vivo and can be harvested in large numbers, from the omentum. Recent work demonstrated that the expression of tissue factor (TF) is induced in MC after isolation and culture. Different culture conditions were studied to suppress TF-expression. MC grown in pooled human serum (HS) are procoagulant (717 +/- 119 pM factor Xa/min.10(5) cells). Replacing HS for fetal calf serum, precoating the surface with extracellular matrix and the addition of the xanthine-oxidase inhibitor allopurinol, inhibited TF expression by 90% (p < 0.001). Allopurinol clearly reduced TF-mRNA levels. TF expression on cultured MC is an in-vitro effect due to culture conditions and the formation of oxygen free radicals. By reducing TF expression by 90%, we have established conditions in which MC are a good alternative for EC for seeding on synthetic grafts.

Base Sequence↗