Protective effect of vitamin E on immune triggered, granulocyte mediated endothelial injury

The effect of alfa-tocopherol on the cell-cell interactions at the vessel wall were studied, using an in vitro model of human umbilical vein endothelial cell cultures (HUEC). Immune triggered granulocytes (PMN) will adhere to and damage HUEC and platelets enhance this PMN mediated endothelial injury. When HUEC are cultured in the presence of vitamin E, 51Cr-leakage induced by complement stimulated PMN is significantly decreased and the enhanced cytotoxicity by platelets is completely abolished (p less than 0.001). The inhibition of PMN induced endothelial injury is directly correlated to a diminished adherence of PMN to vitamin E-cultured HUEC (p less than 0.001), which may be mediated by an increase of both basal and stimulated endogenous prostacyclin (PGI2) from alfa-tocopherol-treated HUEC (p less than 0.025). The vitamin E-effect is abolished by incubation of HUEC with the irreversible cyclo-oxygenase inhibitor, acetylsalicylic acid, but the addition of exogenous PGI2 could not reproduce the vitamin E-mediated effects. We conclude that vitamin E exerts a protective effect on immune triggered endothelial damage, partly by increasing the endogenous anti-oxidant potential, partly by modulating intrinsic endothelial prostaglandin production. The failure to reproduce vitamin E-protection by exogenously added PGI2 may suggest additional, not yet elucidated vitamin E-effects on endothelial metabolism.     

Influence of prostaglandin E2 and indomethacin on interferon-γ production by cultured peripheral blood leukocytes of multiple sclerosis patients and healthy donors

The addition of indomethacin to concanavalin A (Con A)-induced cultures of human peripheral blood leukocytes (PBL) caused an increase in interferon response, regardless of whether the PBLs were derived from multiple sclerosis (MS) patients or from control donors. Specifically the response rates increased from 71 to 100% in controls and from 24 to 53% in MS patient-derived cultures. The amounts of interferon produced also increased in both groups by 0.8 log U/ml. However, interferon yields of nonresponsive cultures becoming interferon-producing only after indomethacin treatment remained relatively low. In control cultures, maximal increases of interferon production were obtained with doses of 0.05 to 0.1 µg/ml indomethacin; for MS patients higher doses were needed—0.1 to 0.5 µg/ml. Conversely, a relatively low dose (0.05 µg/ml) of exogenous prostaglandin E₂ (PGE₂) was able to inhibit interferon production completely in MS patient-derived cultures, whereas in control cultures higher doses were needed (0.1 to 1.0 µg/ml). Analysis of endogenous PGE₂ levels in the PBL cultures revealed that PGE₂ production was similar in nonresponder MS cultures and responder control cultures but that MS leukocytes were more sensitive to the inhibitory effect of PGE₂ on interferon production. We conclude that in a minor percentage of MS patient-derived PBL cultures, the deficiency in interferon- (IFN-) production can be (partially) overcome by treatment of the cells with indomethacin. However, in the major part of nonresponder MS cultures, indomethacin has no effect, indicating that the PG system is not the major cause for the defective interferon response in MS.     

A monoclonal antibody directed against human von Willebrand factor induces type 2B-like alterations.

We have previously described a monoclonal antibody (mAb), 1C1E7, against von Willebrand factor (VWF), that increases ristocetin-induced platelet aggregation (RIPA) and induces a preferential binding of the high-molecular-weight multimers of VWF to platelet GPIb. Further investigations using a rotational viscometer at a shear rate of 4000 s(-1) could now demonstrate that shear-induced platelet aggregation (SIPA) is significantly increased with 1C1E7 and that this could be completely inhibited by the anti-GPIb mAb 6D1. In contrast, platelet adhesion to a collagen surface at a shear rate of 2600 s(-1), using a rectangular perfusion chamber, was significantly inhibited in the presence of 1C1E7. When citrated whole blood was incubated with 1C1E7, a spontaneous binding of VWF to the platelet GPIb could be demonstrated by flow cytometric analysis. Parallel to this, a decrease of the highest molecular weight multimers of VWF in the plasma was found. Platelets with bound VWF on their surface were able to form macroaggregates but were no longer able to adhere. These phenomena are very similar to the alterations described in von Willebrand's disease type 2B. The epitope of this mAb could be localized to the N-terminal part of the subunit; therefore a distant conformational change in the A1 domain of VWF is suggested.     

Inhibitors of the interactions between collagen and its receptors on platelets

At sites of vascular injury, collagen-mediated platelet adhesion and activation have long been known as one of the first events in platelet-dependent thrombus formation. Studying patients with bleeding disorders that are caused by defective platelet adhesion to collagen resulted in the identification of several platelet collagen receptors, with glycoprotein VI and integrin α2β1 being the most important ones. Subsequent development of specific collagen receptor knockout mice and various inhibitors of platelet binding to collagen have further proven the role of these receptors in haemostasis and thrombosis. The search for clinically applicable inhibitors for use as antithrombotic drug has led to the identification of inhibitory antibodies, soluble receptor fragments, peptides, collagen-mimetics and proteins from snake venoms or haematophagous animals. In experimental settings, these inhibitors have a good antithrombotic effect, with little prolongation of bleeding times, suggesting a larger therapeutic window than currently available antiplatelet drugs. However, at present, none of the collagen receptor blockers are in clinical development yet.     

Ridogrel, a combined thromboxane synthase inhibitor and receptor blocker, decreases elevated plasma beta-thromboglobulin levels in patients with documented peripheral arterial disease

The combination of thromboxane synthase inhibition with thromboxane receptor antagonism has been shown to result in a strong inhibition of platelet aggregation and a prolongation of the bleeding time (Gresele et al., J. Clin Invest 1987; 80: 1435-45). Ridogrel is a single molecule that efficiently achieves both inhibitions in human volunteers. The present study was performed in patients with obstructive peripheral arterial disease and elevated plasma beta-thromboglobulin levels. Patients were treated with either 2 x 300 mg ridogrel or 2 x 300 mg placebo per day for 2 1/2 days, according to a double blind randomised parallel design. Plasma beta-thromboglobulin decreased significantly throughout active treatment starting within 2 h after administration; serum and urinary immunoreactive TxB2 levels and urinary 11-dehydro-TxB2 excretion were significantly lower and serum PGE2 and 6-keto-PGF1 alpha levels significantly higher with ridogrel; no changes were observed in the placebo-treated group. In conclusion this study demonstrates a reduction of platelet activation in vivo by ridogrel.     

The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib

The membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.     

Coronary artery in-stent stenosis persists despite inhibition of the von Willebrand factor--collagen interaction in baboons

Revascularization techniques, such as angioplasty and stent implantation, frequently lead to restenosis due to the formation of neointima after platelet activation and the concomitant release of various smooth muscle cell mitogenic and attractant factors. We here investigate whether inhibition of initial platelet adhesion after stent implantation can decrease neointima formation in a clinically relevant baboon model of in-stent stenosis using standard treatment with aspirin, clopidogrel and heparin. Inhibition of platelet adhesion was established by administration of the anti-von Willebrand factor (VWF) monoclonal antibody 82D6A3, which inhibits VWF binding to collagen. Administration of 82D6A3 resulted in a complete inhibition of VWF binding to collagen during the first three days after stent implantation. No thrombocytopenia or prolongation of the bleeding time was observed. Our results show that the formation of neointima was not affected in the group of baboons where primary platelet adhesion was abolished with 82D6A3 when compared to the control group. Vascular injury scores were the same in both groups. Inhibition of platelet adhesion during the first three days after stenting, on top of standard treatment with aspirin, clopidogrel and heparin, had no effect on neo-intima formation in a baboon model of in-stent stenosis. During the last decade, attempts to translate seemingly effective therapies based on smaller animal experimentation to the clinic have consistently failed. This study, using a non-human primate model that more closely resembles the clinical situation, presents a model that may be of further clinical interest for studying the prevention of restenosis.     

Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters

Interaction between exposed collagen and platelets and/or von Willebrand factor is believed to be one of the initiating events for thrombus formation at sites of damaged endothelium. Interference with this mechanism may provide an anti-thrombotic potential. Calin, a product from the saliva of the leech Hirudo medicinalis, was tested in vitro and for its in vivo activity in a thrombosis model in hamsters. Calin specifically and dose dependently (IC50:6.5 to 13 micrograms/mL) inhibited human platelet aggregation induced by collagen. In addition, specific platelet adhesion onto microtiter wells coated with collagen and detected with a monoclonal antiglycoprotein IIb/IIIa antibody- conjugated with horseradish peroxidase, could be completely prevented with Calin (IC50:22 micrograms/mL). A dose-response curve was constructed in groups of six hamsters in whom a standardized trauma was induced on the femoral vein. Thrombus formation was followed continuously using video recording and processing of the image obtained upon transillumination of the vessel. Intravenous Calin dose- dependently inhibited platelet-rich thrombus formation in this model with an ED50 of 0.07 mg/kg and complete inhibition with 0.2 mg/kg. No effects were seen on coagulation tests or bleeding times, whereas ex vivo aggregation induced by collagen was inhibited dose dependently. Local application of leech saliva, Calin, hirudin, or the combination of the latter two into the bleeding time wound of hamsters resulted in a mild prolongation of the bleeding time (twofold to threefold). A similar experiment in baboons did not cause any prolongation of the bleeding time. This is in sharp contrast with the long-lasting bleeding after a leech bite itself in both species. Calin from the leech Hirudo medicinalis is able, by binding to collagen, to effectively interfere with platelet-collagen interaction, which results in an antithrombotic effect observed in a platelet-rich thrombosis model in hamsters.