A Bonnefoy

The evolving role of thrombospondin-1 in hemostasis and vascular biology

Abstract.Thrombospondin-1 (TSP1) is a multi-domain, multi-functional glycoprotein synthesized by many cells. Matricellular TSP1 modulates cell adhesion and proliferation. TSP1 is involved in angiogenesis, inflammation, wound healing and cancer. As a major platelet protein, for a long time it was postulated to control hemostasis via platelet aggregate stabilization. However, these in vitro findings have been questioned in the absence of corroborating clinical data and of obvious hemostatic defects in TSP1 gene-deficient mice.Yet, the past few years have provided indices to implicate TSP1 in hemostasis. In clinical studies, a correlation exists between a welldefined TSP1 polymorphism and a significant risk of myocardial infarction.At the same time, recent in vivo animal model data imply TSP1 in the multimer size control of von Willebrand factor, in smooth muscle cell regulation and in vascular perfusion. These findings shed new light on the role of TSP1 in hemostasis and prothrombotic vascular pathologies. (Part of a Multi-author Review)

Gallic Acid Antagonizes P-Selectin–Mediated Platelet–Leukocyte Interactions Implications for the French Paradox

Background—Current paradigm attributes the low incidence of cardiovascular disorders in Mediterranean countries despite a high saturated fat intake, the “French paradox,” to the antioxidant capacity of red wine polyphenols. Conceivably, other antiinflammatory pathways may contribute to at least a similar extent to the atheroprotective activity of these polyphenols. We have investigated whether gallic acid (GA), an abundant red wine polyphenol, modulates the activity of P-selectin, an adhesion molecule that is critically involved in the recruitment of inflammatory cells to the vessel wall and thus in atherosclerosis. Methods and Results—GA potently inhibited the binding of a peptide antagonist (IC50, 7.2 &mgr;mol/L) and biotin-PAA-Lea-SO3H, an established high-affinity ligand, to P-selectin (IC50, 85 &mgr;mol/L). Under dynamic flow conditions, GA markedly and dose dependently attenuated the rolling of monocytic HL60 cells over P-selectin-transfected Chinese hamster ovary cells (EC50, 14.5 &mgr;mol/L) while increasing the velocity of P-selectin-dependent rolling of human blood leukocytes over a platelet monolayer. In vivo tests established that GA administration to normolipidemic C57/Bl6 and aged atherosclerotic apolipoprotein E–deficient mice impaired the baseline rolling of conjugates between activated platelets and circulating monocytes over femoral vein endothelium, as judged by online video microscopy (ED50, 1.7±0.3 and 1.5±0.4 mg · kg−1 · h−1, respectively). Conclusions—Our findings provide a solid mechanistic foundation through which GA intervenes in major inflammatory pathobiologies by binding and antagonizing P-selectin.

Inhibition of von Willebrand factor-GPIb/IX/V interactions as a strategy to prevent arterial thrombosis.

Although drugs exist for the primary and secondary prevention of thrombosis, more potent antiplatelet drugs with sufficiently wide therapeutic windows to avoid bleeding complications are needed. Both academic and pharmaceutical laboratories are working to develop such drugs. This chapter reviews the potential of inhibiting interactions between von Willebrand factor (vWF) and the second most abundant receptor on the platelet, the glycoprotein (GP) Ib/IX/V complex, interactions that are essential for the activation of circulating platelets, contacting a vessel wall injury. Although still at the level of preclinical testing, this area is expected to progress quickly during the next few years, also in view of the three-dimensional structural information that has recently become available and that allows a molecular understanding of vWF binding to the GPIbα chain of the GPIb complex.

alphaIIbbeta3 antagonism vs. antiadhesive treatment to prevent platelet interactions with vascular subendothelium.

Summary.  Platelets adhering to blood vessels promote coagulation and inflammation, and release growth factors that trigger smooth muscle cell activation. We have therefore studied the pharmacological modification of platelet deposition quantitatively by comparing adhesion of flowing platelets to various subendothelial ligands in the absence or presence of an antiαIIbβ3 antagonist with the effects of antiadhesive treatment consisting of von Willebrand factor (VWF) and fibronectin neutralization or of the combined inhibition of platelet adhesion and aggregation. In vitro, perfusion of anticoagulated human blood over calf skin collagen reiterated that αIIbβ3 antagonism prevents platelet aggregation, but not adhesion per se: single platelets strongly bound to collagen at wall shear rates of both 1300 and 2700 s−1, largely VWF‐independent. When perfused over a human umbilical vein endothelial cell‐derived extracellular matrix, single αIIbβ3‐antagonized platelets primarily adhered to matrix‐bound VWF when perfused at 2700 s−1, but at 1300 s−1 they also adhered significantly to fibronectin. During perfusion of anticoagulated rabbit blood over de‐endothelialized rabbit aorta at a wall shear rate of 1100 s−1, αIIbβ3 antagonism even increased the absolute numbers of adhering platelets and VWF neutralization redirected αIIbβ3‐antagonized platelets towards other vascular ligands. Finally, in vivo, following photochemically induced blood vessel injury in mice, αIIbβ3 antagonism inhibited platelet‐rich thrombus formation, but platelet adhesion was only significantly inhibited when associated with fibronectin neutralization. In conclusion, antiadhesive platelet treatment more potently interferes with platelet deposition on injured blood vessels than αIIbβ3 antagonism, but abrogating platelet adhesion can only be achieved by carefully selected antiplatelet drug combinations.

Thrombospondins: from structure to therapeutics: The evolving role of thrombospondin-1 in hemostasis and vascular biology

Abstract.Thrombospondin-1 (TSP1) is a multi-domain, multi-functional glycoprotein synthesized by many cells. Matricellular TSP1 modulates cell adhesion and proliferation. TSP1 is involved in angiogenesis, inflammation, wound healing and cancer. As a major platelet protein, for a long time it was postulated to control hemostasis via platelet aggregate stabilization. However, these in vitro findings have been questioned in the absence of corroborating clinical data and of obvious hemostatic defects in TSP1 gene-deficient mice.Yet, the past few years have provided indices to implicate TSP1 in hemostasis. In clinical studies, a correlation exists between a welldefined TSP1 polymorphism and a significant risk of myocardial infarction.At the same time, recent in vivo animal model data imply TSP1 in the multimer size control of von Willebrand factor, in smooth muscle cell regulation and in vascular perfusion. These findings shed new light on the role of TSP1 in hemostasis and prothrombotic vascular pathologies. (Part of a Multi-author Review)