ALBERT

Description: Human platelets synthesize and store functionally silent tissue factor (TF) which has procoagulant activity (PCA) after platelet activation. We now explored the location of inactive and active forms of TF and mechanisms of activation. We reported that resting, non-permeabilized human platelets express scant surface TF, which was strikingly enhanced and co-localized with GPIbα after stimulation (Blood2007;109:5242). The externalization of TF was confirmed by immunoprecipitation (Ip) from biotinylated membranes before and after platelet activation. Moreover, TF and GPIbα co-precipitated in Ip assays and both glycoproteins were prominently found in lipid rafts (Lr) domains. TF-dependent PCA, assessed by FXa generation, was negligible or absent in resting, leukocyte-free platelet preparations. Interestingly, FVII was found in platelet membranes (western blot) and no exogenous FVIIa was needed to trigger TF-dependent FXa generation from washed platelets. Platelet responses to activation (TF-dependent PCA, platelet aggregation and secretion) depended on the agonist used. With 1IU/mL VWF + 1.2 mg/mL Ristocetin (Ris) PCA increased 5 to 10-fold 2 min after stimulation and platelets formed large aggregates with null 14C-serotonin secretion. In contrast, both platelet aggregation and secretion were normal 2 min after activation with 1IU thrombin, 10μM TRAP or 2μg/mL collagen; and PCA induced by thrombin was only ≈1/2 of that elicited by VWF+Ris, whereas 2 min after TRAP or collagen stimulation no PCA induction was detected. Removal of membrane cholesterol (by methyl-β-cyclo-dextrin) or disruption of Lr (with filipin III) abolishes the VWF-Ris-induced PCA. Lr’s from resting platelets contain TF of Mr ≈60kDa. Five min after activation an increase in Lr’s TF was observed, but its Mr depended on the agonist: species of ≈47kDa and ≈60kDa were found after VWF-Ris and TRAP activation, respectively. Given that GPIbα signals through Lyn, member of the Src family, inhibition of signaling with PP2 resulted in 80% fall of VWF-Ris-induced PCA. Ip assays revealed that Lyn co-precipitated with both GPIbα and TF in VWF-Ris activated, but not resting platelets. The phosphokinase activity of Lyn on TF was tested. A polyclonal antibody raised against the phosphorylated (Ser253/Ser258) cytoplasmic domain of TF (Dr. W. Ruf, La Jolla) recognized membrane TF only in activated, not in resting platelets. These findings indicate that VWF-induced activation of GPIbα, subsequent signaling with Lyn-induced serine phosphorylation, along with a change of TF to a ≈47kDa species, triggers human platelets PCA. The previously described role of phospho-disulphide isomerases (PDI) in TF activation through SH groups oxidation was also explored. TF and PDI co-precipitated in resting and activated platelet membranes and antagonizing PDI with bacitracin inhibited TF-dependent PCA. After platelet activation and labeling with MPB, a sulfhydryl-specific probe, TF protein (with PCA) was detected on the plasma membrane, denoting presence of reduced thiols. Furthermore, platelet incubation with phenylarsine oxide, a blocking reagent of vicinal SH groups, or HgCl2, a potent oxidant of thiol groups, had no effect on platelet PCA. Thus, it seems unlikely that TF activation depends on SH oxidation. Finally, we found that platelet TF was sufficient to speed up the clotting of plasma. In fact, clotting time of PRP (2 × 108 platelets mL−1) incubated with ionophore A23187 (2 min, 37°C) and then re-calcified, was 59 ± 6 sec, whereas clotting time in re-calcified PRP without previous activation was 137 ± 19 sec (n=19, p

Description: Abstract 4205 Background. Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We have previously shown (Pereira et al. J Thromb Haemost 2009; 7[suppl 2]: 232a) that chronic cocaine use is associated with markers of endothelial dysfunction and platelet activation in subjects studied after recent consumption. It has been suggested that many of the adverse effects of cocaine on the vessel wall are due to its acute effects related to the sympathomimetic properties of the drug. However, from a pathogenic standpoint, we hypothesized that important pathologic consequences, such as early onset atherosclerosis and regional brain perfusion, defects, are not solely explained by the acute vasomotor actions of cocaine. Objectives. The main aim of this work was to investigate the effect of short-term abstinence on markers of endothelial injury and platelet activation in chronic cocaine consumers. methods . We studied 23 cocaine dependent individuals (aged 19–52years mean age 30 years) who met DSM-IV criteria for cocaine dependence, seeking treatment for cocaine abuse and 25 healthy controls (aged 20–49 years, mean age 31 years). Samples were obtained at admission within 72 hours of drug exposure and after 4 weeks of strict, controlled abstinence in a rehabilitation clinic. Endothelial cell damage was assessed by enumerating circulating endothelial cells (CECs) and plasma levels of soluble markers: soluble intercellular adhesion molecule (sICAM); monocyte chemoattractant protein (MCP-1), von Willebrand factor (VWF) and high-sensitivity C reactive protein (hs-CRP). Plasma levels of soluble CD40L (sCD40L), NAP-2 and RANTES were determined to demonstrate platelet activation in vivo. Results. Markers of endothelial cell damage/activation and platelet activation, were significantly higher in cocaine dependents individuals after recent consumption (baseline) as compared with the controls. The variations observed after 4 weeks drug withdrawal (post-abstinence) are shown in Table 1: Conclusions. Our results demonstrate that cocaine use is associated to endothelial dysfunction and platelet activation which are prominent findings after recent consumption. Evidence of endothelial cell activation/damage is still present after 4 weeks of strict and controlled abstinence when markers of platelet activation returned to their baseline levels. Taken together, these observations suggest that cocaine induced-endothelial cell damage is maintained independent of the acute effect of the drug on the blood vessels. The persistence of this condition may play a role in long-term ischemic complications associated with cocaine abuse such as early onset atherosclerosis and regional brain perfusion defects. Further studies on the mechanisms underlying cocaine-induced endothelial dysfunction might provide novel strategies to improve endothelial function as part of the treatment in recently abstinent cocaine addicts. Disclosures: No relevant conflicts of interest to declare.

Description: Acute inflammation in response to severe bacterial infection, results in hemostatic abnormalities ranging from subclinical to sustained systemic clotting activation leading to massive thrombin and fibrin formation and microvascular thrombosis. Endothelial activation and dysfunction are critical determinants of the host response and provide an explanation for the different abnormalities involved in the pathophysiology of sepsis. Infection with pathogenic E. coli may present with a wide spectrum of clinical manifestations, from no symptoms or mild non-bloody diarrhea to severe cases, such as hemolytic uremic syndrome or thrombotic thrombocytopenic purpura, which is characterized by hemolytic anemia and low platelet counts. Although the understanding of the mechanisms that are involved in blood coagulation abnormalities in sepsis has gradually progressed, the role of platelets (Plts) on the procoagulant state during a severe infection remains to be addressed. Human platelets contain functional tissue factor (TF) (Panes et al. 2007) and TFPIa, but it is unknown if bacteria-platelet interaction affects platelet-TF procoagulant or platelet TFPI anticoagulant activities. Moreover, the effects of bacterial activation of platelets on thrombin generation (TG) in platelet rich plasma (PRP) or adhesion to endothelial cells have not been explored. Aims: We assessed the effect of platelet-E. coli interaction on platelet TF-dependent procoagulant activity (PCA), the changes induced by this interaction on platelet TFPI, in TG in PRP and in the adhesive capacity of platelets on cultured HUVEC. Plts activation by E. coli was demonstrated by a significant increase of p-selectin exposure on platelet surface compared to control Plts after 30 min of interaction with this microorganism harvested at exponential phase of growth and incubated in a ratio Plts/bacteria 1:10. Platelet TF-dependent PCA was assessed by FXa generation in washed Plts exposed to E. coli, with addition of exogenous FVIIa and FX, with no extra source of TF. Using the same ratio Plts/bacteria, we observed an increase in FXa after 30 min of incubation, compared with control platelets (p=0.0002, n=12). This enhancement in TF-PCA was concomitant with a decreased expression of TFPI in Plts surface after exposure of PRP to E. coli for 30 min. (p=0.0012, n=9). TG was measured in PRP, previously stimulated by E. coli for 30 min. We observed a shortening in the Lag time and time to peak and a higher thrombin peak in stimulated than in control PRP (p=0.0001, p=0.005 and p=0.0342, respectively, n=12). The reduction in lag time and time to peak was more pronounced than that obtained after eliciting platelet activation with Ristocetin. Preincubation of Plts with E. coli also increased the velocity index of TG compared to PRP alone (p=0,005; n=12). Static adhesion of Pts to endothelium was studied by stimulating fresh washed Plts with E. coli for 30min and then co-incubating them with HUVEC. After 20 min, an increased number of bacterial-activated Plts were adhered to HUVEC, compared with unstimulated Pts. Moreover, visible Plts aggregates were observed, which were positive for fibrin immunostaining, suggesting clot formation during the interaction of Plts with E. coli O111. Our findings show that Plts activated by bacteria results in an enhanced platelet procoagulant activity and adhesion to endothelium. By extension, these in vitro results suggest that platelets play an important role in the prothrombotic state associated with bacterial infections. This work was supported by FONDECYT-Chile 1130835 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 156 Human platelets store TF that expresses procoagulant activity (PCA) associated with platelet activation (Panes O. et al, Blood 2007;109:5242-50). We have also observed that platelet TF-induced PCA is specifically and rapidly induced by activation of GPIb complex by VWF-Ristocetin (Panes O. et al. Blood 2008;112:A113). Hypercholesterolemia has been associated with increased platelet function and hypercoagulability, but the mechanisms are still unknown. The cholesterol content in cell membranes is directly related with the plasma concentration of the sterol. Inhibition of HMG-CoA reductase by statins reduces cardiovascular risk by decreases in plasma cholesterol and by other independent (“pleiotropic”) effects. We studied the relationship between total and LDL plasma cholesterol with platelet TF content and TF-dependent PCA of washed platelets activated for 5 min with VWF-Ristocetin (VWF-R). We also examined these variables after atorvastatin or rosuvastatin intake (80 and 20 mg/day × 1 month, respectively) in 25 subjects with hypercholesterolemia. TF was measured by ELISA and PCA by FXa generation (chromogenic assay) in washed platelet suspensions before and after activation. We found no significant differences between both statins regarding the decrease in plasma LDL-cholesterol levels and platelet TF-dependent PCA; both statins had any significant effects on the levels of blood inflammatory markers (usCRP, fibrinogen and VWF). Thus, we analyzed the pooled data of patients receiving either one or the other statin. Total and LDL-Chol dropped from 271±41 and 185±40 to 167±30 and 87±23 mg dL-1, respectively. This was associated with decrease in basal PCA (non-stimulated platelets) from 34±27 to 22±14 nmol of FXa/2*107platelets (p=0.02). After VWF-R stimulation, a mean 3.48-fold increase in platelet PCA was observed in hypercholesterolemic patients before taking statins. Surprisingly, a mean 20-fold increase in FXa generation in activated platelets was observed after 1 month on statin therapy, and this difference was statistically significant (p=0.018). These changes in PCA were not associated with significant changes in TF content of platelet membranes (621±481 to 555±300 pg/mg prot). We also found that plasma LDL-Chol was negatively correlated with platelet PCA induced by VWF-R activation (r = −0.239, p = 0.026) and with the ratio of PCA between activated and non-activated platelets (r = −0.27, p = 0.011). The striking post-statin increase of platelet-PCA without change in the total platelet TF protein suggests that PCA depends more on the fraction of platelet TF available for activation than on the total mass of platelet TF. The decrease in basal PCA of non-stimulated platelets after statin therapy suggests that lower concentration of cholesterol in platelet membranes makes the platelets more stable during the isolation procedure. The higher increase after stimulation would reflect a better hemostatic response to a specific stimulus. Thus, changes in plasma cholesterol, possibly through decrease in platelet membrane cholesterol, modulates the PCA of platelets, either by making platelets more stable under resting conditions and more responsive after specific activation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1116 Platelets are intrinsic components of hemostatic and pathological clots, and are essential for clot retraction. However, their role and sequential involvement in clot stabilization and lysis are still poorly understood. Human platelets contain several components of the fibrinolytic system, including functional PAI-1, TAFI, uPA and α 2-antiplasmin. Moreover, platelets possess a rich transcriptome and synthesize several proteins, among them, PAI-1. Using a global, modified clot lysis time assay in platelet-rich plasma (CLT-PRP; Panes et al., Platelets 2012) we found that the CLT-PRP was significantly longer than that of CLT in platelet-free plasma (PFP), reflecting a down-regulation of the fibrinolytic process. However, the prolonged CLT in subjects receiving tranexamic acid was normalized earlier in PRP than in PPP, denoting some pro-fibrinolytic activity in clots formed in a platelet milieu. Aim: to study the presence, origin, association and functional role of components of the fibrinolytic system in human platelets. Also, we aim to getting insight into the dynamic balance and modulation of the fibrinolytic process by the interplay of pro- and anti-fibrinolytic platelet factors. Methods and Results: in washed, leukocyte-free human platelets we detected expression of LRP-1, uPAR, PAI-1 mRNAs, and synthesis of these proteins (metabolic radiolabeling). Neither uPA mRNA nor synthesis of uPA was evidenced. All of these proteins, including uPA were detected in membrane or cytosol fractions by western blotting (WB). LRP-1 and uPAR were present in the outer leaflet of platelet membranes, with increased uPAR labeling after platelet activation (confocal microscopy-immunofluorescence). Non-stimulated whole platelets exhibit a low basal uPA activity (specific chromogenic substrate) selectively inhibited by amiloride. uPA activity falls slightly immediately after VWF-Ristocetin (VWF-R) and TRAP stimulation, but recovers to basal levels after 15min. Biotinylated washed platelets were immunoprecipitated (IP) with α -uPAR MoAb at different times before and after activation with either TRAP or VWF-Ristocetin. Co-precipitations with LRP-1, PAI-1 and uPA were detected in WB only after platelet activation with TRAP for 5 min, denoting the formation of a tetrameric complex, likely involved in endocytosis and receptor recycling. Interestingly, 5min after TRAP stimulation, uPA was sharply reduced, disappearing at 15 min, either in membrane or cytosol fractions, suggesting degradation of the protein. Similar pattern of co-precipitations were observed when IP was done with α -LRP-1 MoAb. Co-precipitations were more prominent in purified platelet membrane than in cytosolic fractions. Conclusions: human platelets express LRP-1, uPAR and PAI-1 mRNAs, and synthesize these proteins. uPA activity is present in whole, purified, washed platelets, and the protein is likely bound to the external platelet membrane. Co-precipitation of all these fibrinolytic components presumably denotes the formation of a tetrameric complex with endocytic and recycling capacities, as demonstrated in other cell lineages. Sequential IP′s after platelet activation disclose the disappearance of uPA, but not of PAI-1, from the complex, probably explained by a degradation process. Taken together, these results suggest that platelets play a predominantly antifibrinolytic role during early stages of formation of platelet-rich clots. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3302 Glycoprotein VI (GPVI) is a trans-membrane collagen receptor expressed in blood platelets and megakaryocytes. Collagen, a major extracellular matrix constituent is a distinctive platelet ligand that mediates platelet adhesion and triggers platelet activation. Thus, GPVI has a central role in platelet adhesion and signal transduction. GPVI is a 64kDa member of the immunoglobulin receptor family, with an extracellular portion of 247 Aa harboring two immunoglobulin-type domains, followed by a mucin-like, presumably O-glycosylated Ser-Thr-rich region, a short peptide linker sequence, a 21 Aa trans-membrane domain and a short 51 Aa cytoplasmic tail. This receptor is also activated by laminin, collagen-related peptide (CRP) and snake toxins, i.e., convulxin and alborrhagin. Inherited defects of major platelet GP′s, e.g, αIIbβ3 integrin or GPIb-IX-V complex cause severe disorders of primary hemostasis, namely Glanzmann thrombasthenia and Bernard-Soulier syndrome, respectively. Another collagen receptor in platelets is integrin α2β1, with scarce reports of molecular defects clinically expressed as mild bleeding disorder. Acquired platelet GPVI defects, characterized by absent platelet aggregation with collagen, have been described in patients with immune thrombocytopenia, in whom autoantibodies are directed against GPVI causing shedding of the receptor from the platelet membrane. Two recent reports have described single patients with abnormal bleeding associated with compound heterozygous mutations of GPVI. In the first case, the patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. GPVI DNA sequencing of the second patient also showed 2 genetic abnormalities, an R38C mutation in exon 3 of one allele and an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Now we report 4 unrelated Chilean patients living in different places along the country, with a unique GPVI mutation clinically manifested as mild or moderate bleeding disorders. All the patients suffered from mucous and skin bleeding and one of them had received platelet transfusions to treat heavy menorrhagias. All these patients had null platelet aggregation and 14C-serotonin secretion when stimulated with collagen types I and II, convulxin and collagen-related peptides (CRP). Platelet responses to arachidonate and ADP were normal, and 2 of these patients had only first-wave aggregation with epinephrine. The mutation in these patients consists in an homozygous adenine insertion at position 712 of the cDNA, which causes a translation frameshift, creating a premature stop codon which predicts a truncated form of the protein. The mutation detected in the patient's gDNA was reflected in their platelet mRNA. The sequencing of the RT-PCR products obtained from platelets of homozygous carriers of the mutation showed the adenine insertion as well. Heterozygous relatives had no bleeding disorder, and normal aggregation and 14C-serotonin secretion with collagen and convulxin, but decreased aggregation with CRP. Flow cytometry and immunofluorescence-confocal microscopy revealed absence of membrane GPVI in the 4 homozygous carriers of the mutation, whereas heterozygous relatives exhibited a decreased expression of the GP on their platelets. Antibodies recognizing the extracellular portion of the receptor were used for Western blots of platelet membrane fractions or platelet lysates. Blots showed a single band of approximately 49 KDa in patients, whereas heterozygous relatives expressed both the normal 64kDa integral receptor and the abnormal protein band. The size of the truncated protein was that predicted by the mutation. This is the first report describing a point mutation in the GPVI gene with functional and clinical consequences. The collection of 4 of these non-related patients suggests that this defect may be more common than previously suspected. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. In this sense, in vitro studies have demonstrated that cocaine may induce damage and/or activation of endothelial cells. The structural and functional integrity of the endothelium is essential for the maintenance of vascular homeostasis and its damage plays a critical role in the pathogenesis of vascular disease. Endothelial dysfunction may be assessed by testing the impaired vasodilator response to a stimulus or by measuring the release of plasma markers of endothelial damage. Increased number of circulating endothelial cells (CECs) has been reported in several pathologic conditions associated with severe vascular damage and its enumeration in peripheral blood is currently considered a reliable method to assess endothelial damage. We hypothesized that chronic exposure to cocaine induces endothelial damage which could be expressed by increased CEC counts in peripheral blood. Methods: Twenty cocaine-dependent subjects (aged 19–52 years, mean age 30 years) and 25 healthy, matched controls (aged 20–49 years, mean age 31 years) were studied; all patients fulfilled DSM-IV criteria for cocaine dependence with drug exposure within the 72 hours prior to blood sampling. CECs were isolated from peripheral blood by use of immunomagnetic beads coated with anti-CD146, stained for CD45 and Ulex Europaeus lectin 1 and counted under fluorescence microscopy. MCP-1, sICAM-1, von Willebrand factor and high-sensitivity C-reactive protein (hsCRP) were measured by enzyme-linked immunosorbent assay. Results: Compared to controls, CECs were significantly elevated among cocaine users (632 ± 281 vs 67 ± 54 cells/mL, p

Description: The binding of platelet membrane GPIb-IX-V to extracellular matrix proteins and von Willebrand factor initiates the adhesion, spreading and activation of platelets. On the other hand, tissue factor (TF) triggers the blood coagulation by binding FVII/VIIa. In resting platelets, TF is stored mainly as a non active (“encrypted”) form, possibly through a dimer- or heterodimer complex. The encryption seems not to affect the TF capacity to bind FVII and VIIa, in such form that the membrane-based complex would be “primed” to trigger the clot formation upon platelet activation. Accordingly, the platelet adhesion and activation would be tightly interwoven with thrombin generation. Given the described localization of GPIbα in platelet lipid rafts, which appears to modulate its activity, we investigated whether TF is also present in these cholesterol-rich domains of the platelet membrane. Continuous sucrose gradients generated by ultracentrifugation at 180.000g x 18h at 4°C were used to obtain cholesterol-rich fractions from platelet lysates (1% Triton X-100 at 4°C). Western blots of each gradient fraction using a polyclonal anti-TF antibody revealed a protein of ~60kDa only in the fractions rich in cholesterol (using flotillin-1 as a marker). TF-dependent procoagulant activity (PCA), assessed by FXa generation was measured in resting and 5μM TRAP-stimulated, leukocyte-free platelets. In both cases, PCA was reduced after membrane cholesterol depletion with methyl-β-cyclo-dextrin. We also found that PCA was significantly enhanced in washed platelets treated with VWF and ristocetin, suggesting some form of functional or structural relationship between GPIb-IX-V complex and TF. GPIb-IX was immunoprecipitated (IP) from human platelet membranes by anti-GPIbα MoAbs (AP-1 or SZ2), in both resting and stimulated platelets (TRAP or VWF-ristocetin). The IP product was revealed with MoAb anti-TF (American Diagnostica, clone 4509) or polyclonal anti-TF, detecting a protein of ~47kDa. When we used MoAb anti-TF for the IP and MoAb anti-GPIX (BL-H6, anti-CD42a) to reveal the western blot, two distinct bands of ~22 and ~47kDa were disclosed, probably corresponding to GPIX and a heterodimer of GPIX and TF, respectively. These observations indicate that, at least a fraction of the platelet TF is localized in lipid rafts and that TF is also in close association with the GPIb-IX-V complex. This co-localization probably has important functional implications as demonstrated by the defective thrombin generation in patients with Bernard-Soulier syndrome. Moreover, this functional association may be relevant in both normal hemostasis and thrombus formation and growth.