Platelet factor-4 (PF4/CXCL4) is an orphan chemokine released in large quantities in the vicinity of growing blood clots. Coagulation of plasma supplemented with a matching amount of PF4 results in a translucent jelly-like clot. Saturating amounts of PF4 reduce the porosity of the fibrin network 4.4-fold and decrease the values of the elastic and loss moduli by 31-and 59-fold, respectively. PF4 alters neither the cleavage of fibrinogen by thrombin nor the cross-linking of protofibrils by activated factor XIII but binds to fibrin and dramatically transforms the structure of the ensuing network. Scanning electron microscopy showed that PF4 gives rise to a previously unreported pattern of polymerization where fibrin assembles to form a sealed network. The subunits constituting PF4 form a tetrahedron having at its corners a RPRH motif that mimics (in reverse orientation) the Gly-His-Arg-Pro-amide peptides that co-crystallize with fibrin. Molecular modeling showed that PF4 could be docked to fibrin with remarkable complementarities and absence of steric clashes, allowing the assembly of irregular polymers. Consistent with this hypothesis, as little as 50 M the QVRPRHIT peptide derived from PF4 affects the polymerization of fibrin.In addition to catalyzing the conversion of fibrinogen to fibrin, thrombin activates the G protein-coupled protease-activated receptor 1, triggering platelet adhesion and activation (1, 2). Following activation, large quantities of platelet factor-4 (PF4/CXCL4), 3 a major component of the ␣-granules (15-20 g/10 9 platelets), are released into the vicinity of growing blood clots, such that amounts in excess of 5 g/ml are found in serum (3, 4). PF4 is an asymmetrically associated homo-tetrameric (70 residues/subunit) chemokine that interacts with a variant of CXCR3. The physiologic function of PF4 is not fully understood even if one of the first clues to the existence of endogenous angiogenesis inhibitors came with the observation that it inhibits endothelial cell proliferation (5). PF4 is otherwise mainly known for binding polysulfated glycosaminoglycans such as heparin and has been extensively studied for its role in heparin-induced thrombocytopenia (6). Generally speaking, PF4 binds to glycosaminoglycans, modulating their activity. Both pro-coagulant and anti-coagulant properties for PF4 have been reported. PF4 accelerates the activation of the anticoagulant protein C (7, 8); in contrast, studies on transgenic mice demonstrate that whereas PF4 contributes to thrombus formation, PF4-null mice had no overt bleeding diathesis (9).Fibrinogen is a M r 340,000 glycoprotein consisting of a pair of ␣-, ␤-, and ␥-chains. The molecule comprises two outer D-nodules connected through coiled-coil domains to a central E-nodule (10, 11). The E-nodule includes all six NH 2 termini, and each D-nodule contains the COOH terminus of the ␤-and ␥-chains folded into globular domains, namely ␤C and ␥C (12, 13). Fibrin formation is initiated by a thrombin-catalyzed removal of two peptides (fibrinopeptides A) from t...

ObjectiveIn acute cerebral ischemia, circulating neutrophil count and neutrophil-to-lymphocyte ratio (NLR) are positively associated with stroke severity and worse outcomes. Mediators of this effect are unknown. We aimed to investigate (1) the relationship between plasma matrix metalloproteinase-9 (MMP-9) and myeloperoxidase (MPO) concentrations with stroke severity and outcome and (2) MMP-9 and MPO release after ex vivo stimulation of neutrophils by recombinant tissue plasminogen activator (rtPA).MethodsWe analyzed data collected in 255 patients with supratentorial cerebral infarcts recruited within 48 hours of symptoms onset irrespective of rtPA treatment. The endpoints were excellent outcome (modified Rankin Scale score 0–1), symptomatic intracerebral hemorrhage (European Cooperative Acute Stroke Study–II definition), and death at 3 months. The role of rtPA treatment on peripheral neutrophil degranulation was investigated in 18 patients within 4.5 hours and after 72 hours.ResultsNeutrophil counts, NLR, and MPO plasma concentrations, but not MMP-9, were positively correlated with stroke severity. Higher neutrophil counts and NLR were independently associated with worse outcomes and higher mortality rates at month 3. Higher MPO plasma concentrations, but not MMP-9, were associated with worse outcome. Neutrophil-derived MMP-9, after ex vivo rtPA stimulation, but not MPO, were higher after 72 hours in patients treated by IV rtPA but not associated with hemorrhagic transformation.ConclusionsNeutrophil counts, NLR, and MPO plasma concentrations are associated with worse outcome in patients with acute cerebral ischemia, in contrast to MMP-9. Further investigations are needed to deepen our knowledge on MPO’s role in the deleterious effect of neutrophils because it could represent a potential therapeutic target.

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BackgroundGranulocyte colony-stimulating factor (G-CSF) is a pharmacologic agent inducing neutrophil mobilization and a new candidate for neuroprotection and neuroregeneration in stroke. Its effects when used in combination with tissue plasminogen activator (tPA) were explored during the acute phase of ischemic stroke.MethodsWe used a middle cerebral artery occlusion (MCAO) model of cerebral ischemia, associated with treatment with tPA, in male spontaneously hypertensive rats (SHR). Granulocyte colony-stimulating factor (G-CSF; 60 μg/kg) was injected just before tPA. Neutrophil response in peripheral blood and in the infarct area was quantified in parallel to the infarct volume. Protease matrix metallopeptidase 9 (MMP-9) release from circulating neutrophils was analyzed by immunochemistry and zymography. Vascular reactivity and hemorrhagic volume in the infarct area was also assessed.ResultsTwenty four hours after ischemia and tPA, G-CSF administration induced a significant increase of neutrophils in peripheral blood (P <0.05). At 72 hours post-ischemia, G-CSF was significantly associated with an increased risk of hemorrhage in the infarct area (2.5 times more likely; P <0.05) and significant cerebral endothelium-dependent dysfunction. Ex vivo, an increased MMP-9 release from neutrophils after tPA administration correlated to the increased hemorrhagic risk (P <0.05). In parallel, G-CSF administration was associated with a decreased neutrophil infiltration in the infarct area (-50%; P <0.05), with a concomitant significant neuroprotective effect (infarct volume: -40%; P <0.05).ConclusionsWe demonstrate that G-CSF potentiates the risk of hemorrhage in experimental stroke when used in combination with tPA by inducing neutrophilia. This effect is concomitant to an increased MMP-9 release from peripheral neutrophils induced by the tPA treatment. These results highlight the potential hemorrhagic risk of associating G-CSF to thrombolysis during the acute phase of stroke.

Classical hemophilia results from a defect of the intrinsic tenase complex, the main factor X (FX) activator. Binding of factor VIIa to tissue factor triggers coagulation, but little amplification of thrombin production occurs. Handling of hemophilia by injection of the deficient or missing (thus foreign) factor often causes immunological complications. Several strategies have been designed to bypass intrinsic tenase complex, but none induce true auto-amplification of thrombin production. In an attempt to re-establish a cyclic amplification of prothrombin activation in the absence of tenase, we prepared a chimera of FX having fibrinopeptide A for the activation domain (FX FpA ). We reasoned that cascade initiation would produce traces of thrombin that would activate FX FpA (contrary to its normal homologue). Given that the activation domain of FX is released upon activation, thrombin cleavage would produce authentic FXa that would produce more thrombin, which in turn would activate more chimeras. Blood clotting results from a cascade of zymogen activation that culminates with a large production of thrombin (1-4). Injury initiates the process by uncovering tissue factor (TF), 3 which captures circulating activated factor VII (FVIIa) to form a complex that activates factors IX (FIX) and X (FX). Traces of activated FX (FXa) are sufficient to produce a small amount of thrombin empowering the cascade to inflate. The two main amplification complexes within this cascade are intrinsic tenase, composed of activated factor VIII (FVIIIa) associated with FIXa, and prothrombinase, composed of activated factor V (FVa) associated with FXa. The former activates FX into FXa, the latter, prothrombin into thrombin. Hemophilias A and B are coagulopathies resulting from a dysfunction of the intrinsic tenase complex, following a defect of FVIII or FIX, respectively (5). As a consequence, there is an inadequate production of FXa and subsequently of thrombin. Despite normal triggering, little amplification of thrombin production occurs. To date, replacement therapy is the only treatment that re-establishes an intrinsic auto-amplification of thrombin production (6). Therapy simply consists of administering the deficient FVIII or FIX. The main drawback of this approach resides in the potential antigenicity of the injected molecule, because it is often seen as foreign by the recipient. Neutralizing allo-antibodies makes replacement therapy gradually ineffective. Three approaches that bypass the intrinsic tenase complex without re-establishing auto-amplification of thrombin formation have been used or envisaged. In the first, injection of a mixture of the vitamin K-dependent factors (FVII, FIX, FX, and prothrombin) effectively bypasses tenase. Treatment however induces rare but serious side effects, including anaphylactic shock and thrombotic incidents. A mixture of recombinant prothrombin and FXa is currently under consideration to improve this approach (7). An efficient alternative relies on supraphysiologic injection of FVIIa (8 -9). Un...

This study aimed to investigate atherosclerotic mediators' expression levels in M1 and M2 macrophages and to focus on the influence of diabetes on M1/M2 profiles. Macrophages from 36 atherosclerotic patients (19 diabetics and 17 non-diabetics) were cultured with interleukin-1β (IL-1β) or IL-4 to induce M1 or M2 phenotype, respectively. The atherosclerotic mediators' expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that M1 and M2 macrophages differentially expressed mediators involved in proteolysis and angiogenesis processes. The proteolytic balance (matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-9/plasminogen activator inhibitor-1 (PAI-1) and MMP-9/tissue factor pathway inhibitor-2 (TFPI-2) ratios) was higher in M1 versus M2, whereas M2 macrophages presented higher angiogenesis properties (increased vascular endothelial growth factor/TFPI-2 and tissue factor/TFPI-2 ratios). Moreover, M1 macrophages from diabetics displayed more important proangiogenic and proteolytic activities than non-diabetics. This study reveals that M1 and M2 macrophages could differentially modulate major atherosclerosis-related pathological processes. Moreover, M1 macrophages from diabetics display a deleterious phenotype that could explain the higher plaque vulnerability observed in these subjects.

The genomic CDKN2A/B locus, encoding p16INK4a among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16INK4a is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16INK4a knock-down in 3T3/L1 preadipocytes or p16INK4a deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16INK4a in adipose tissue formation. p16INK4a-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16INK4a-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16INK4a-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16INK4a gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16INK4a modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

Taken together, these findings indicate that leptin plays a critical role in CAVD development by promoting osteoblast differentiation of human aortic VICs in an Akt- and ERK-dependent manner. This study highlights the role of leptin in CAVD development, and further studies are needed to determine whether reducing circulating leptin levels or blocking leptin actions on VICs is efficient to slow CAVD progression.