H. J. M. Brinkman

Ionizing radiation enhances platelet adhesion to the extracellular matrix of human endothelial cells by an increase in the release of von Willebrand factor.

The effect of radiation on the secretion of von Willebrand factor by endothelial cells was studied in a three-compartment culture system. The release of von Willebrand factor was significantly increased at 48 h after a single gamma-radiation dose of 20 Gy in both the luminal and abluminal direction by 23 (P < 0.05) and 41% (P < 0.02), respectively. To establish whether the enhanced production of von Willebrand factor affected the thrombogenicity of the extracellular matrix, platelet adhesion to the matrix produced by a monolayer of cultured endothelial cells during 48 h after irradiation was analyzed in a perfusion chamber at high shear rate (1300 s-1). Platelet adhesion was significantly increased by irradiation both in the presence and in the absence of plasmatic von Willebrand factor by 65 (P < 0.05) and 34.5% (P < 0.005), respectively. Incubation of the perfusate with a monoclonal antibody that blocks the binding of von Willebrand factor to platelet GPIb (CLB-RAg 35) resulted in an almost complete inhibition of platelet adhesion. These data indicate that radiation enhances platelet adhesion to the the extracellular matrix by an increase in the release of von Willebrand factor by endothelial cells. This event may be important in early radiation-induced vascular pathology.

Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.

To study the biology of the endothelium under conditions that mimic the architecture of the vessel wall, endothelial cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cultures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter.

In vitro assessment, using thrombin generation, of the applicability of prothrombin complex concentrate as an antidote for Rivaroxaban

Rivaroxaban has been approved as an antithrombotic agent for prevention of venous thromboembolism with specific indications. At present no antidote is appointed and no guidelines have been formulated for the measurement of Rivaroxaban reversal.

The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes

SUMMARY. Rates of factor X activation on endothelial cells were compared with activation rates on other vascular cells, platelets, monocytes and negatively charged phospholipid vesicles. Factor VIIa‐mediated factor X activation was observed on smooth muscle cells and fibroblasts in the absence of cell‐perturbing agents, whereas endothelial cells required activation in order to allow extrinsic activation of factor X. On the other hand, unperturbed endothelial cells did promote intrinsic, factor VIII/IXa‐dependent activation of factor X. The rate of factor X activation on these cells was about one‐sixth of that on ionophore A23187‐stimulated platelets. Also, smooth muscle cells and fibroblasts were able to activate factor X through the intrinsic pathway, altough to a lesser extent than endothelial cells. Monocytes were ineffective in this respect. Prothrombin fragment 1, the prothrombin fragment containing the γ‐carboxyglutamic acid domain known to mediate binding of vitamin K‐dependent coagulation factors to phospholipid surfaces, inhibited factor VIII/IXa‐dependent factor X activation on endothelial cells (IC50 3.2 μM) to a lesser extent than on phospholipid vesicles (IC50 0.2 μM). Therefore, besides negatively charged phospholipids, other membrane constituents seem to be involved in endothelial cell mediated, intrinsic activation of factor X. Perturbation of endothelial cells with phorbol myristate acetate (PMA) or lipopoly‐saccharide (LPS) was without effect on intrinsic activation of factor X. This observation indicates that membrane constituents of endothelial cells involved in factor VIII/IXa‐dependent activation of factor‐X are constitutively expressed.

Polar secretion of von Willebrand factor by endothelial cells

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the polarity of von Willebrand factor (vWF) secretion. Endothelial cells cultured under these conditions allow direct measurements of substances released at both the apical and basolateral surface. The constitutive secretion of vWF was compared to the release of vWF from their storage granules after stimulation (regulated secretion). The basal, constitutive release of vWF occurs into both the apical and subendothelial direction. The rate of accumulation of vWF to the subendothelial direction is about three times higher than the amount of vWF secreted into the lumenal medium per unit of time. However, upon stimulation of confluent endothelial cell monolayers with phorbol myristate acetate, endothelial cells predominantly secrete vWF at the lumenal surface. Under these conditions, vWF does not accumulate in the collagen matrix. Thus, endothelial cells are able to organize themselves into a polarized monolayer, in such a way that vWF secreted by the regulated pathway accumulates at the lumenal site, whereas resting endothelial cells release vWF predominantly at the opposite, basolateral surface.

Von Willebrand factor targets IL-8 to Weibel-Palade bodies in an endothelial cell line.

Vascular endothelial cells are able to store the chemotactic cytokine interleukin-8 (IL-8) in specialized storage vesicles, Weibel-Palade bodies, together with von Willebrand factor (VWF) and P-selectin. We investigated whether VWF plays a role in the sorting of IL-8 into these organelles. We examined the effect of VWF expression on IL-8 targeting in an endothelial cell line (EC-RF24). This cell line has retained the typical phenotypic characteristics of primary endothelial cells but has lost the capacity to produce VWF in appreciable amounts. EC-RF24 cells were retrovirally transduced with a vector encoding a VWF-green fluorescent protein chimera (VWF-GFP). This approach enables direct visualization of the cellular distribution and secretory behavior of the VWF-GFP hybrid. Expression of VWF-GFP resulted in the generation of Weibel-Palade body-like organelles as shown by the colocalization of VWF-GFP and P-selectin. VWF-GFP expressing EC-RF24 cells also showed significant colocalization of VWF-GFP with IL-8 in these storage vesicles. Live cell imaging revealed that the number of VWF-GFP-containing granules decreased upon cell stimulation. These observations indicate that VWF plays an active role in sequestering IL-8 into Weibel-Palade bodies.

The platelet glycoprotein Ia-IIa-associated Br-alloantigen system is expressed by cultured endothelial cells

Summary. To obtain information on the immunological relationship between the endothelial and platelet glycoprotein (GP)Ia‐IIa (VLA‐2) complex, we studied whether endothelial GPIa‐IIa was able to express the platelet GPIa‐IIa‐associated Br‐alloantigen system. Therefore, we tested antisera to both allelic forms of the Br system (Bra and Brb) on platelets (by an assay based on monoclonal antibody‐specific immobilization of platelet antigens, MAIPA) and on cultured umbilical vein endothelial cells (by immunoprecipitation experiments) from the same individual. Endothelial cells from a platelet Br(a + b +), and from a platelet Br(a ‐ b +) individual were studied. Our results indicate that endothelial GPIa‐IIa is indistinguishable from platelet GPIa‐IIa in its ability to express the Bra and Brb alloantigens. The association of Br alloantigens with endothelial GPIa‐IIa was confirmed by the results of an assay based on monoclonal antibody‐specific immobilization of endothelial antigens (MAIEA). These data further illustrate the structural and immunologic similarity of platelet and endothelial cell GPIa‐IIa (VLA‐2).

Factor seven activating protease (FSAP): does it activate factor VII?

Background: Factor seven activating protease (FSAP) was initially reported as an activator of single‐chain urokinase‐type plasminogen activator (scuPA) and factor VII (FVII). Subsequently, numerous additional substrates have been identified, and multiple other biological effects have been reported. Due to the apparent lack of specificity, the physiological role of FSAP has become increasingly unclear. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources. Objectives: Our aim was to produce intact recombinant human FSAP, and to assess its role as a trigger of coagulation and fibrinolysis. Results: Expression of wild‐type FSAP in various mammalian cells invariably resulted in the accumulation of degraded FSAP due to autoactivation and degradation. To overcome this problem, we constructed a variant in which Arg313 at the natural activation site was replaced by Gln, creating a cleavage site for the bacterial protease thermolysin. HEK293 cells produced FSAPR313Q in its intact form. Thermolysin‐activated FSAP displayed the same reactivity toward the substrate S‐2288 as plasma‐derived FSAP, and retained its ability to activate scuPA. Polyphosphate and heparin increased Vmax by 2–3‐fold, without affecting Km (62 nm) of scuPA activation. Surprisingly, FVII activation by activated FSAP proved negligible, even in the presence of calcium ions, phospholipid vesicles and recombinant soluble tissue factor. On membranes of 100% cardiolipin FVII cleavage did occur, but this resulted in transient activation and rapid degradation. Conclusions: While FSAP indeed activates scuPA, FVII appears remarkably resistant to activation. Therefore, reappraisal of the putative role of FSAP in hemostasis seems appropriate.

Selective conversion and esterification of monohydroxyeicosatetraenoic acids by human vascular smooth muscle cells: Relevance to smooth muscle cell proliferation

5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE), lipoxygenase metabolites of arachidonic acid that may modulate cell proliferation, were examined for their ability to affect the [3H]thymidine incorporation of human umbilical artery smooth muscle cells. We found that these hydroxy fatty acids inhibited the serum-induced [3H]thymidine incorporation of growth-arrested vascular smooth muscle cells in a similar dose-dependent manner. The inhibitory effect was dependent on the serum concentration used to stimulate cell growth. The higher the serum concentration, the lower the inhibitory effect of the HETE. In parallel experiments, the incorporation of HETEs into lipids of the smooth muscle cells was examined. After 20 h of incubation, we found that in the presence of 0.4% serum 70% of 3H-labeled 5-HETE was esterified into human vascular smooth muscle cell lipids. Twelve and eight percent, respectively, of 12- and 15-HETE were incorporated into smooth muscle cell lipids. Furthermore, we found that during the 20-h incubation of human umbilical artery smooth muscle cells with 12- and 15-HETE, these compounds were converted into metabolites with a chromatographic behavior on HPLC similar to that of diHETEs. 5-HETE was not converted into these polar metabolites. Increasing the serum concentration resulted in a decreased metabolism of all HETEs tested. Thus, the distinct differences between the metabolism of different HETEs by vascular smooth muscle cells does not reflect the proliferation inhibitory effect of these HETEs.

Proteolytic cleavage of protein S during the hemostatic response

Summary.  Background: Protein S is a vitamin K‐dependent protein with anticoagulant properties. It contains a so‐called thrombin‐sensitive region (TSR), which is susceptible to cleavage by coagulation factor Xa (FXa) and thrombin. Upon cleavage, the anticoagulant activity of protein S is abolished. Objective: The aim of the present study was to determine whether protein S is cleaved within the TSR during activation of the coagulation system under near physiological conditions. Results: In a reconstituted coagulation system containing apart from protein S only procoagulant constituents and synthetic phospholipid vesicles, protein S was cleaved at Arg60 by the FXa generated (3 mol min−1 mol−1 enzyme). FXa‐catalyzed cleavage of protein S, however, was inhibited by factor Va and prothrombin by more than 70%. During clotting of recalcified citrated plasma in the presence of a synthetic lipid membrane, no FXa‐catalyzed proteolysis of protein S was observed. Substituting platelets for phospholipid vesicles resulted both in the reconstituted system and in plasma in cleavage of the TSR. Cleavage was at Arg60 and was observed upon platelet activation, irrespective of the presence of FXa (13 pmol min−1 10−8 platelets). No cleavage by thrombin was observed in either the reconstituted coagulation system or clotting plasma. Conclusion: These findings suggest that in vivo the anticoagulant activity of protein S is not down‐regulated by FXa or thrombin during activation of coagulation. Our results rather suggest a role for a platelet protease in down‐regulating the anticoagulant activity of protein S during the hemostatic response.