Jan A. van Mourik

Real-Time Imaging of the Dynamics and Secretory Behavior of Weibel-Palade Bodies

Objective—Weibel-Palade bodies (WPBs) are specialized secretory granules found in endothelial cells. These vesicles store hormones, enzymes, and receptors and exhibit regulated exocytosis on cellular stimulation. Here we have directly visualized intracellular trafficking and the secretory behavior of WPBs in living cells by using a hybrid protein consisting of von Willebrand factor (vWF), a prominent WPB constituent, and green fluorescent protein (GFP). Methods and Results—Immunofluorescence microscopy demonstrated that this chimera was targeted into WPBs. In resting cells, some WPBs seemed motionless, whereas others moved at low speed in a stochastic manner. On stimulation of cells with [Ca2+]i- or cAMP-raising secretagogues, membrane-apposed patches were formed, suggesting fusion of WPBs with the plasma membrane. Patches remained visible for >20 minutes. This sustained, membrane-associated retention of vWF might play a role in focal adhesion of blood constituents to the endothelium after vascular injury. In addition, stimulation with cAMP-raising agonists resulted in clustering of a subset of WPBs in the perinuclear region of the cell. Apparently, these WPBs escaped secretion. This feature might provide a mechanism to control regulated exocytosis. Conclusions—In conclusion, the fusion protein vWF-GFP provides a powerful tool to study, in real time, signal-mediated trafficking of WPBs.

Small GTP-Binding Protein Ral Modulates Regulated Exocytosis of von Willebrand Factor by Endothelial Cells

Abstract—Weibel-Palade bodies are endothelial cell–specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.

Effect of the Binding of Anti‐Zwa Antibodies on Platelet Function

Abstract. Anti‐Zwa antibodies can induce a Glanzmann‐like platelet dysfunction in normal Zwa‐positive platelets. Although a normal shape change and release reaction was always recorded in response to adenosine diphosphate and collagen, the aggregation on these inducers could be completely inhibited by anti‐Zwa antibodies. Analogous to the platelets from patients with Glanzmanns thrombasthenia, normal platelets sensitized with anti‐Zwa did not associate with fibrinogen upon exposure to adenosine diphosphate. Our data indicate that the binding site for fibrinogen is closely associated with the Zwa‐antigenic determinants on the platelet membrane glycoproteins and thus with glycoprotein IIIa which is known to carry Zwa.

Dynein-Dynactin Complex Mediates Protein Kinase A-Dependent Clustering of Weibel-Palade Bodies in Endothelial Cells

Objective—Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell–specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail. Methods and Results—Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein–dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering. Conclusions—These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein–dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein–dynactin complex.

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.

Correlation between the potency of a beta2-glycoprotein I-dependent lupus anticoagulant and the level of resistance to activated protein C

The antiphospholipid syndrome is characterized by the occurrence of vascular thrombosis combined with the presence of antiphospholipid antibodies in plasma of patients. It has been published that antibeta2-glycoprotein I (beta2-GPI) antibodies, with lupus anticoagulant activity (LAC), highly correlate with thrombosis. Resistance related to antiphospholipid antibodies against activated protein C (APC) is one of the proposed mechanisms responsible for thrombosis. We investigated a possible correlation between a beta2-GPI-dependent LAC (titration of cardiolipin into an activated partial thromboplastin time-based assay) and increased APC resistance in a population of 22 plasma samples with LAC activity. Eleven plasma samples that displayed a beta2-GPI-dependent LAC also showed increased APC resistance. In contrast, only one of the 11 plasma samples with a beta2-GPI-independent LAC displayed increased APC resistance. In addition, a monoclonal antibeta2-GPI antibody and patient-purified immunoglobulin G (both with LAC activity) were diluted in plasma with/without protein C. Both antibodies only displayed a beta2-GPI-dependent LAC in plasma in the presence of protein C. This indicates that the principle of the beta2-GPI LAC-assay was based on increased resistance against protein C. Surface plasmon resonance analysis was used to investigate binding between APC and beta2-GPI. We observed that beta2-GPI was able to bind APC directly, especially in the presence of a monoclonal antibeta2-GPI antibody. In conclusion, our observations show a direct correlation between a major clinical symptom of antiphospholipid syndrome (thrombosis), a diagnostic assay (beta2-GPI-dependent LAC) and a potential mechanism responsible for thrombosis in the antiphospholipid syndrome (increased APC resistance).

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).

Endothelial cell activation in thrombotic thrombocytopenic purpura (TTP): a prospective analysis

Summary. It is generally assumed that endothelial cell injury is the primary event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In this study, we have determined the extent of vascular perturbation during acute episodes of the disease. We performed a prospective, serial study of nine patients with relapsing TTP during hospitalization and treatment, and assessed the degree of endothelial cell involvement at admission, exacerbation and remission by measurement of von Willebrand factor (VWF) and VWF‐propeptide levels. Measurement of both VWF and its propeptide enabled discrimination between acute and chronic perturbation of the endothelium. Elevated levels of both VWF and propeptide were found at admission. These levels decreased immediately upon plasma exchange therapy. However, plasma VWF and propeptide concentrations did not change, even at the time of acute exacerbation. These observations suggest that endothelial cell activation is not the primary event leading to TTP. Vascular perturbation seems to be a consequence, rather than a cause, of the disease.

Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann's thrombasthenia

A patient is described with a disturbance of platelet function comparable to that in Glanzmanns thrombasthenia. Platelet aggregation and binding of fibrinogen to the patients platelets were defective and thrombin‐induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced. As measured by monoclonal antibody binding in the presence of divalent cations, the platelets contained about 15% of the normal amount of GPIIb and GPIIIa and only 6% of the normal amount of intact GPIIb/IIIa complex. The residual GPIIb/IIIa complex exhibited a decreased stability as shown by the lack of binding of a complex‐dependent anti‐GPIIb/IIIa antibody to platelets incubated with ethylene diamine tetraacetic acid (EDTA) at 22°C. Crossed immunoelectrophoresis (CIE) in the presence of divalent cations showed partial dissociation of GPIIb/IIIa as well as the presence of two forms of the residual intact GPIIb/IIIa complex. In addition, both CIE in the presence of the EDTA and two‐dimensional sodium dodecyl sulphate (SDS) gel electrophoresis showed the presence of two forms of GPIIb. This form of thrombasthenia is characterized by a defective platelet function, a marked reduction of GPIIb and GPIIIa, decreased stability of the residual GPIIb/IIIa complex and structural heterogeneity of GPIIb.

Persistent factor VIII-dependent factor X activation on endothelial cells is independent of von Willebrand factor

Endothelial cells are able to support the activation of coagulation factor X by activated factor IX in the presence of its cofactor, factor VIII. We have previously reported that this reaction is persistent on endothelial cells, but transient on activated platelets and phospholipid vesicles when activated factor X (Xa) is used as activator of factor VIII. Aim of the present study was to explore the influence of von Willebrand factor and that of the factor VIII activator, either factor Xa or thrombin, on the decay of factor X activation on the endothelial cell surface. Kinetics of factor X activation on human umbilical vein endothelial cells was compared with that on phospholipid vesicles employing purified coagulation factors from plasma as well as recombinant factor VIII variants. Employing factor Xa as factor VIII activator, rate constants for decay of membrane-bound factor X activation were consistently low on endothelial cells (0.02 min−1) as compared with phospholipid vesicles (0.2 min−1). Activation of factor VIII by thrombin resulted in two-fold increased decay rates. In the presence of excess of von Willebrand factor over factor VIII, decay rates were not significantly changed. Factor VIII variants with and without a Tyr1680 to Phe substitution, which abolishes high-affinity binding to von Willebrand factor, displayed the same factor X activation decay kinetics. Although previous studies have shown that von Willebrand factor modulates factor VIII activation and stabilisation, this apparently does not affect the progression of factor X activation at the endothelium.