URL: http://www.clinicaltrials.gov. Unique identifier: NCT02907736.

A cute ischemic stroke (AIS) remains a leading cause of disability, cognitive impairment, and mortality worldwide, despite the development of revascularization therapies. Besides recanalization status, other prognostic factors in AIS patients are associated with clinical outcome. Among them, hyperglycemia has been found to be associated with hemorrhagic transformation (HT) and worsened neurological outcomes. 1,2 Given that 40% to 50% of AIS patients present with hyperglycemia, 3 understanding and reducing hyperglycemia-induced neurovascular injury constitute an important clinical stake. Experimental studies have investigated extensively the relationship between hyperglycemia and poor outcome after AIS. [4][5][6] Notably, these studies have shown that preexisting hyperglycemia, whether acute or prolonged, was an important determinant of brain injury in AIS. In contrast, hyperglycemia induced after the period of ischemia had no effect on experimental stroke outcome.5 Hyperglycemia was further shown to increase cerebral ischemia-reperfusion-induced blood-brain Background and Purpose-Admission hyperglycemia is associated with a poor outcome in acute ischemic stroke. How hyperglycemia impacts the pathophysiology of acute ischemic stroke remains largely unknown. We investigated how preexisting hyperglycemia increases ischemia/reperfusion cerebral injury. Methods-Normoglycemic and streptozotocin-treated hyperglycemic rats were subjected to transient middle cerebral artery occlusion. Infarct growth and brain perfusion were assessed by magnetic resonance imaging. Markers of platelet, coagulation, and neutrophil activation were measured in brain homogenates and plasma. Downstream microvascular thromboinflammation (DMT) was investigated by intravital microscopy. Results-Hyperglycemic rats had an increased infarct volume with an increased blood-brain barrier disruption and hemorrhagic transformation rate compared with normoglycemic rats. Magnetic resonance imaging scans revealed that hyperglycemia enhanced and accelerated lesion growth and was associated with hemorrhagic transformation originating from territories that were still not completely reperfused at 1 hour after middle cerebral artery recanalization. Intravital microscopy and analysis of brain homogenates showed that DMT began immediately after middle cerebral artery occlusion and was exacerbated by hyperglycemia. Measurement of plasma serotonin and matrix metalloproteinase-9 indicated that platelets and neutrophils were preactivated in hyperglycemic rats. Neutrophils from hyperglycemic diabetic patients showed increased adhesion to endothelial cells as compared with neutrophils from normoglycemic donors in flow chamber experiments. Conclusions-We show that hyperglycemia primes the thromboinflammatory cascade, thus, amplifying middle cerebral artery occlusion-induced DMT. DMT exacerbation in hyperglycemic rats impaired reperfusion and precipitated neurovascular damage, blood-brain barrier disruption, and hemorrhagic transformation. Our results designate DMT as a po...

A high plasma concentration of lipoprotein Lp(a) is now considered to be a major and independent risk factor for cerebro‐ and cardiovascular atherothrombosis. The mechanism by which Lp(a) may favour this pathological state may be related to its particular structure, a plasminogen‐like glycoprotein, apo(a), that is disulfide linked to the apo B100 of an atherogenic LDL‐like particle. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen‐like kringle 4 and single copies of kringle 5 and the catalytic region. At least one of the plasminogen‐like kringle 4 copies present in apo(a) (kringle IV type 10) contains a lysine binding site (LBS) that is similar to that of plasminogen. This structure allows binding of these proteins to fibrin and cell membranes. Plasminogen thus bound is cleaved at Arg561‐Val562 by plasminogen activators and transformed into plasmin. This mechanism ensures fibrinolysis and pericellular proteolysis. In apo(a) a Ser‐Ile substitution at the Arg‐Val plasminogen activation cleavage site prevents its transformation into a plasmin‐like enzyme. Because of this structural/functional homology and enzymatic difference, Lp(a) may compete with plasminogen for binding to lysine residues and impair, thereby, fibrinolysis and pericellular proteolysis. High concentrations of Lp(a) in plasma may, therefore, represent a potential source of antifibrinolytic activity. Indeed, we have recently shown that during the course of the nephrotic syndrome the amount of plasminogen bound and plasmin formed at the surface of fibrin are directly related to in vivo variations in the circulating concentration of Lp(a) (Arterioscler. Thromb. Vasc. Biol., 2000, 20: 575–584; Thromb. Hæmost., 1999, 82: 121–127). This antifibrinolytic effect is primarily defined by the size of the apo(a) polymorphs, which show heterogeneity in their fibrin‐binding activity—only small size isoforms display high affinity binding to fibrin (Biochemistry, 1995, 34: 13353–13358). Thus, in heterozygous subjects the amount of Lp(a) or plasminogen bound to fibrin is a function of the affinity of each of the apo(a) isoforms and of their concentration relative to each other and to plasminogen. The real risk factor is, therefore, the Lp(a) subpopulation with high affinity for fibrin. According to this concept, some Lp(a) phenotypes may not be related to atherothrombosis and, therefore, high Lp(a) in some individuals might not represent a risk factor for cardiovascular disease. In agreement with these data, it has been recently reported that Lp(a) particles containing low molecular mass apo(a) emerged as one of the leading risk conditions in advanced stenotic atherosclerosis (Circulation, 1999, 100: 1154–1160). The predictive value of high Lp(a) as a risk factor, therefore, depends on the relative concentration of Lp(a) particles containing small apo(a) isoforms with the highest affinity for fibrin. Within this context, the development of agents able to selectively neutralise the antifibrinolytic activity of Lp(a), ...

High plasma levels of lipoprotein(a) [Lp(a)] are considered to be an independent risk factor for premature cardiovascular disease and are inversely associated with apolipoprotein(a) [apo(a)] isoform sizes. The contribution of apo(a) polymorphism to the inhibition of fibrinolysis, a mechanism that may favor thrombus development, was therefore evaluated by measuring the ability of Lp(a) particles of distinct apo(a) isoform content to compete with plasminogen for fibrin binding during plasminogen activation by fibrin-bound tissue-type plasminogen activator. The rate of plasmin generation was most efficiently inhibited by an isoform with a molecular weight (M(r)) of approximately 540 Kd. An isoform with M(r) approximately 590 Kd produced a less pronounced effect, whereas the isoform with M(r) approximately 610 Kd failed to inhibit plasminogen activation. These effects were directly proportional to the amount of Lp(a) bound to the carboxy-terminal lysine residues of degraded fibrin. The relative affinity of the binding (kd range, 16 to 180 nmol/L) reflected the ability of individual Lp(a) isoforms to inhibit the binding of plasminogen (kd, 600 nmol/L). The question of the influence of kringle sequence variability on the binding to fibrin was not addressed by the present work. These data suggest that apo(a) isoform types with high affinity for fibrin may influence the ability of Lp(a) to interfere with fibrinolysis and contribute thereby to the association of elevated levels of Lp(a) with atherosclerotic and thrombotic risks.

SummaryThe interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 ± 46 molec./platelet, Kd = 5.6 ± 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction.These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

SummaryThe fibrino(geno)lytic protein designated hementerin contained in crude extracts of the salivary complex of Haementeria depressa leeches was purified to apparent homogeneity by gel filtration, ion exchange chromatography and preparative SDS-PAGE. It is a single-chain 80 kDa, PhMeSO2F-resistant, calcium-dependent, metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin, another fibrino(geno)lytic protein purified from Haementeria ghilianii leeches. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by hementerin of fibrinogen Aα, γ and Bβ chains, in that order, produces 270 kDa to 67 kDa fragments which differ from those produced by plasmin. Hementerin was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. In conclusion, hementerin is a plasminogen-independent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents blood coagulation and destroys fibrin clots in vitro.

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Protease nexin-1 (PN-1) is a non-circulating pericellular serpin expressed by vascular cells. PN-1 inhibits different proteases but when associated with glycosaminoglycans, its activity is mainly directed towards thrombin. Fucoidans are sulphated polysaccharides which can interact with several serpins and have antithrombotic and anticoagulant properties in vivo with a lower hemorrhagic risk than heparin. The purpose of this study was to compare the effects of low (LMW) or high molecular weight (HMW) fucoidans to those of standard heparin and LMW heparin on PN-1 properties. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and affinity coelectrophoresis, we observed that polysaccharides bound to thrombin, PN-1 and the thrombin/PN-1 complex. Progress curve kinetics showed that LMW and HMW fucoidans accelerate thrombin inhibition by PN-1 (111 and 402 fold, respectively) whereas the acceleration by LMW heparin and standard heparin was only of 36- and of 307-fold, respectively. Moreover, the formation of PN-1/(125)I-thrombin complex was increased in the presence of heparin, HMW and LMW fucoidans, but barely by LMW heparin. The dose response followed a bell shape curve, again suggesting the formation of ternary complexes between thrombin, PN-1 and polysaccharides. We also investigated the ability of polysaccharides to remove PN-1 bound to the cell membrane of smooth muscle cells in culture. PN-1 was detached by fucoidans and heparins and was still able to inhibit thrombin. In conclusion, fucoidans reduce cell-associated PN-1 and thrombin/PN-1 complexes and increase the antithrombin activity of PN-1. The capacity of PN-1 to regulate the pericellular activity of thrombin amongst other proteases reinforces the therapeutical interest of fucoidans.