Effect of the small molecule plasminogen activator inhibitor‐1 (PAI‐1) inhibitor,PAI‐749, in clinical models of fibrinolysis

Summary. Background: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor‐1 (PAI‐1). PAI‐749 is a small molecule inhibitor of PAI‐1 with proven antithrombotic efficacy in several preclinical models. Objective: To assess the effect of PAI‐749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma‐based and whole blood‐based models of fibrinolysis. Methods: In a double‐blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue‐type plasminogen activator (t‐PA) in the presence of PAI‐749 or control. t‐PA‐mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. Results: There was a dose‐dependent reduction in ex vivo thrombus formation by t‐PA (P < 0.0001). PAI‐749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t‐PA. Inhibition of PAI‐1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). Conclusions: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI‐749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood‐based systems.     

Plasminogen on the surfaces of fibrin clots prevents adhesion of leukocytes and platelets

Summary. Background and Objectives: Although leukocytes and platelets adhere to fibrin with alacrity in vitro, these cells do not readily accumulate on the surfaces of fibrin clots in vivo. The difference in the capacity of blood cell integrins to adhere to fibrin in vivo and in vitro is striking and implies the existence of a physiologic antiadhesive mechanism. The surfaces of fibrin clots in the circulation are continually exposed to plasma proteins, several of which can bind fibrin and influence cell adhesion. Recently, we have demonstrated that adsorption of soluble fibrinogen on the surface of a fibrin clot results in its deposition as a soft multilayer matrix, which prevents attachment of blood cells. In the present study, we demonstrate that another plasma protein, plasminogen, which is known to accumulate in the superficial layer of fibrin, exerts an antiadhesive effect. Results: After being coated with plasminogen, the surfaces of fibrin clots became essentially non‐adhesive for U937 monocytic cells, blood monocytes, and platelets. The data revealed that activation of fibrin‐bound plasminogen by the plasminogen‐activating system assembled on adherent cells resulted in the generation of plasmin, which decomposed the superficial fibrin layer, resulting in cell detachment under flow. The surfaces generated after the initial cell adhesion remained non‐adhesive for subsequent attachment of leukocytes and platelets. Conclusion: We propose that the limited degradation of fibrin by plasmin generated by adherent cells loosens the fibers on the clot surface, producing a mechanically unstable substrate that is unable to support firm integrin‐mediated cell adhesion.     

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo

Summary. Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot‐bound FXa and clot‐bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non‐specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC₅₀) of Xa and IIa activity were, respectively: heparin 120 ± 7 and 3 ± 1 ng mL^?1; SanOrg123781A 77 ± 5 and 4 ± 1 ng mL^?1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC₅₀ values for inhibition of FXa and FIIa activity were, respectively: heparin 100 ± 5 and 800 ± 40 ng mL^?1; SanOrg123781A 10 ± 5 and 30 ± 3 ng mL^?1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot‐bound FXa and to some extent to clot‐bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot‐bound FXa with IC₅₀ values of 2 ± 0.5 µg mL^?1 and 0.6 ± 0.2 µg mL^?1, respectively. Both compounds also inhibited clot‐bound thrombin activity, the IC₅₀ values of heparin and SanOrg123781A being 1 ± 0.01 µg mL^?1 and 0.1 ± 0.1 µg mL^?1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot‐bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot‐bound FXa with IC₅₀ values of 4 ± 0.6 and 1 ± 0.1 µg mL^?1, respectively. As with clot‐bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre‐existing thrombus and as activators of recombinant tissue‐type plasminogen activator‐induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot‐bound enzymes and in in vivo models of thrombosis/thrombolysis.     

Neuroprotective effects of ultra‐low‐molecular‐weight heparin in vitro and vivo models of ischemic injury

This study was conducted to demonstrate ultra‐low‐molecular‐weight heparin’s neuroprotective effects on ischemic injury both in vivo and in vitro studies. In vitro, the effect of ultra‐low‐molecular‐weight heparin was tested in cultured PC12 cells exposed to Earle’s solution containing sodium dithionite, to identify its neuroprotection to PC12 cells damaged by oxygen‐glucose deprivation (OGD). The cell injury was detected by the tetrazolium salt 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5 diphenyl‐2H tetrazolium bromide (MTT) assay. In vivo, male Wistar rats with middle cerebral artery occlusion were evaluated for infarct volume followed by the treatment with ultra‐low‐molecular‐weight heparin. The results in vitro showed that ultra‐low‐molecular‐weight heparin significantly inhibited PC12 cells damage induced by OGD. Results in vivo showed that vein injection of Ultra‐Low‐molecular‐weight heparin at doses of 0.5 and 1.0 mg/kg exerted significant neuroprotective effects on rats with focal cerebral ischemic injury by significantly reducing the infarct volume compared with the injury group. All the findings suggest that ultra‐low‐molecular‐weight heparin might act as a neuroprotective agent useful in the treatment of cerebral ischemia.     

Heparin‐induced thrombocytopenia – therapeutic concentrations of danaparoid,unlike fondaparinux and direct thrombin inhibitors,inhibit formation of platelet factor 4–heparin complexes

Summary. Background: Treatment of heparin‐induced thrombocytopenia (HIT), a disorder in which anti‐platelet factor 4 (PF4)–heparin antibodies cause platelet activation and hypercoagulability, requires alternative (non‐heparin) anticoagulation. Treatment options include direct thrombin inhibitors [lepirudin and argatroban (approved), and bivalirudin], danaparoid (approved) (mixture of anticoagulant glycosaminoglycans), or fondaparinux (synthetic heparin‐mimicking pentasaccharide). PF4–heparin complexes form at optimal stoichiometric ratios. Objectives: To compare the effects of these various non‐heparin anticoagulants in disrupting the formation of PF4–heparin complexes, and PF4‐containing immune complexes. Patients/methods: Sera were obtained from patients with serologically confirmed HIT. The effects of the alternative anticoagulants on PF4 and PF4–heparin complex interactions with platelets, as well as HIT antibody binding and platelet activation, were investigated. Results: Danaparoid at very low concentrations increased PF4 binding to platelets. In therapeutic concentrations, however, it decreased PF4 binding to platelets (P = 0.0004), displaced PF4–heparin complexes from platelets (P = 0.0033) and PF4 from the surface of a PF4‐transfected HEK‐293 EBNA cell line expressing the PF4 receptor CXCR3‐B (P = 0.0408), reduced PF4–heparin complex size (P = 0.025), inhibited HIT antibody binding to PF4–heparin complexes (P = 0.001), and prevented platelet activation by HIT antibodies (P = 0.046). Although fondaparinux also interfered with PF4 binding to platelets, HIT antibody binding to PF4–heparin complexes, and activation of platelets by HIT antibodies, these effects occurred only at supratherapeutic concentrations. The direct thrombin inhibitors had no effect at any concentrations. Conclusions: Danaparoid uniquely interferes with the pathogenesis of HIT by disrupting PF4‐containing immune complexes at therapeutic dose concentrations. It is possible that these effects contribute to its therapeutic efficacy.     

Redundant functions of phospholipases D1 and D2 in platelet α‐granule release

Summary. Background: Platelet activation and aggregation are crucial for primary hemostasis, but can also result in occlusive thrombus formation. Agonist‐induced platelet activation involves different signaling pathways leading to the activation of phospholipases, which produce second messengers. The role of phospholipase C (PLC) in platelet activation is well established, but less is known about the relevance of phospholipase D (PLD) . Objective and methods: The aim of this study was to determine a potential function of PLD2 in platelet physiology. Thus, we investigated the function of PLD2 in platelet signaling and thrombus formation, by generating mice lacking PLD2 or both PLD1 and PLD2. Adhesion, activation and aggregation of PLD‐deficient platelets were analyzed in vitro and in vivo. Results: Whereas the absence of PLD2 resulted in reduced PLD activity in platelets, it had no detectable effect on the function of the cells in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective α‐granule release and protection in a model of FeCl₃‐induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. Conclusion: These results reveal redundant roles of PLD1 and PLD2 in platelet α‐granule secretion, and indicate that this may be relevant for pathologic thrombus formation.     

Variability of anti-PF4/heparin antibody results obtained by the rapid testing system ID-H/PF4-PaGIA

Summary. Background: Recent studies have shown that a low clinical pretest probability may be adequate for excluding heparin‐induced thrombocytopenia. However, for patients with intermediate or high pretest probability, laboratory testing is essential for confirming or refuting the diagnosis. Rapid assessment of anti‐PF4/heparin‐antibodies may assist clinical decision‐making. Objectives: To evaluate the performance of rapid ID‐H/PF4‐PaGIA. In particular, we verified reproducibility of results between plasma and serum specimens, between fresh and frozen samples, and between different ID‐H/PF4‐polymer lots (polystyrene beads coated with heparin/PF4‐complexes). Patients/Methods: The samples studied were 1376 plasma and 914 corresponding serum samples from patients investigated for suspected heparin‐induced thrombocytopenia between January 2000 and October 2008. Anti‐PF4/heparin‐antibodies were assessed by ID‐H/PF4‐PaGIA, commercially available ELISAs and heparin‐induced platelet aggregation test. Results: Among 914 paired plasma/serum samples we noted discordant results (negative vs. low‐titre positive) in nine instances (1%; 95%CI, 0.4–1.6%). Overall, agreement between titres assessed in plasma vs. serum was highly significant (Spearman correlation coefficient, 0.975; P < 0.0001). Forty‐seven samples tested both fresh and after freezing/thawing showed a good agreement, with one discordant positive/negative result (Spearman correlation coefficient, 0.970; P < 0.0001). Among 1376 plasma samples we noted a strikingly variable incidence of false negative results (none – 82%; 95%CI, 66–98%), depending on the employed ID‐H/PF4‐polymer lot. Faulty lots can be recognized by titrating commercial positive controls and stored samples of HIT‐patients. Conclusion: Laboratories performing the assay should implement stringent internal quality controls in order to recognize potentially faulty ID‐H/PF4‐polymer lots, thus avoiding false negative results.     

Low molecular weight heparin inhibits plasma thrombin generation via direct targeting of factor IXa: contribution of the serpin‐independent mechanism

Summary. Background: Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined. Objectives: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin‐independent inhibition of intrinsic tenase (FIXa–FVIIIa) activity. Patients/Methods: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC₅₀s) of low molecular weight heparin (LMWH), super‐sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX‐dependent and FIX‐independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock‐depleted or antithrombin/FIX‐depleted plasma supplemented with recombinant FIX. Results: UFH and fondaparinux demonstrated similar potency under FIX‐dependent and FIX‐independent conditions, whereas LMWH (2.9‐fold) and ssLMWH (5.1‐fold) demonstrated increased potency with limiting TF. UFH (62‐fold) and fondaparinux (42‐fold) demonstrated markedly increased EC₅₀ values in antithrombin‐depleted plasma, whereas LMWH (9.4‐fold) and ssLMWH (two‐fold) were less affected, with an EC₅₀ within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin‐depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin‐binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition. Conclusions: Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin‐independent interaction with the FIXa heparin‐binding exosite.     

Polyphosphate binds with high affinity to exosite II of thrombin

Summary. Background: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin. Objective: To examine the interaction of polyphosphate with thrombin. Methods and Results: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin’s C‐terminal dodecapeptide and γ‐thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na⁺‐binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (K_d approximately 5 nm ) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin‐catalyzed inactivation of thrombin by antithrombin. Conclusion: Polyphosphate interacts with thrombin’s exosite II at a site that partially overlaps with, but is not identical to, the heparin‐binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nm K_d for the polyphosphate–thrombin interaction.     

Expression of EMMPRIN (CD147) on circulating platelets in vivo

Summary. Background and Objectives: EMMPRIN (CD147) is a matrix metalloproteinase inducer present on leukocytes and recently identified on platelets in vitro. We examined platelet CD147 expression in vivo and in correlation with markers of platelet activation and coronary artery disease (CAD). Patients/Methods: This prospective observational study involved 70 subjects (55 patients with CAD and 15 controls). Platelet CD62P expression, PAC‐1 expression, platelet–leukocyte aggregates and CD147 (both platelet and leukocyte) expression were assessed by flow cytometry, and soluble CD62P expression was assessed by enzyme‐linked immunosorbent assay. A full blood count and high‐sensitivity C‐reactive protein test were performed. Results: CD147 was expressed on 20.45% ± 1.63% (mean ± standard error of the mean) of circulating platelets, whereas CD62P and PAC‐1 were expressed on 0.87% ± 0.12% and 0.90% ± 0.27% of platelets, respectively. Platelet CD147 expression correlated with CD62P expression (r = 0.359, P = 0.002), PAC‐1 expression (r = 0.428, P < 0.001), leukocyte CD147 expression (monocyte, r = 0.416, P = 0.001; granulocyte, r = 0.434, P < 0.001), C‐reactive protein level and neutrophil/lymphocyte ratio (NLR). CAD patients had significantly higher CD147 mean fluorescence intensity than controls on circulating platelets (2.41 ± 0.14 vs. 2.87 ± 0.09, P = 0.014), monocytes (8.57 ± 1.20 vs. 12.3 ± 0.57, P = 0.006) and granulocytes (4.30 ± 0.65 vs. 6.50 ± 0.34, P = 0.005). Age adjustment eliminated the association between platelet CD147 expression and CAD, but the association between leukocyte CD147 expression and CAD persisted. According to multivariate analysis, the independent predictors of platelet CD147 expression were monocyte CD147 expression, NLR and age. Conclusions: Platelet CD147 expression is evident in vivo and correlates moderately with traditional platelet activation markers and leukocyte CD147 expression. Platelet CD147 expression shows a stronger association with age, and leukocyte CD147 expression a stronger association with clinical CAD, suggesting differences in the regulation of platelet and leukocyte CD147 expression in vivo.     

Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells

Summary. Background: Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described. Objectives: To anchor three serpins –α₁‐proteinase inhibitor (α₁PI) (M358R), antithrombin (AT), and heparin cofactor II (HCII) – in the plasma membranes of transfected mammalian cells and assess their ability to inhibit thrombin. Methods: Serpin cDNAs were altered to include N‐terminal, non‐cleavable plasma membrane‐targeting sequences from the human transferrin receptor (TR) (TR‐serpin) or the human asialoglycoprotein receptor (AR) (AR‐serpin), and used to transfect COS‐1 or HEK 293 cells. Cells were analyzed for serpin expression by immunoblotting of subcellular fractions, by immunofluorescence microscopy, or by flow cytometry, with or without exposure to exogenous thrombin; AR‐serpins and TR‐serpins were also compared with their soluble recombinant counterparts. Results: Both TR‐α₁PI (M358R) and AR‐α₁PI (M358R) were enriched in the integral membrane fraction of transfected COS‐1 or HEK 293 cells, and formed inhibitory complexes with thrombin, although less rapidly than soluble α₁PI (M358R). Thrombin inhibition was abrogated by an additional T345R mutation in AR‐α₁PI (M358R). Surface‐displayed AR‐AT also formed serpin–enzyme complexes with thrombin, but to a lesser extent than AR‐α₁PI (M358R); AR‐HCII inhibitory function was not detected. Immunofluorescence detection and flow cytometric quantification of bound thrombin also supported the status of AR‐α₁PI (M358R) and AR‐AT as thrombin inhibitors. Conclusions: Two of three thrombin‐inhibitory serpins retained functionality when expressed as integral membrane proteins. Our findings could be applied to create and screen hypervariable serpin libraries expressed in mammalian cells, or to confer protease resistance on engineered cells in vivo.     

Platelet Gs hypofunction and abnormal morphology resulting from a heterozygous RGS2 mutation

Summary. Background: Regulator of G‐protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. Objective: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. Methods: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild‐type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. Results: The mutation leads to reduced cAMP production after stimulation of Gs‐coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild‐type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal α‐granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. Conclusions: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.     

Reversible biotinylated oligosaccharides: a new approach for a better management of anticoagulant therapy

Summary. Objective: In order to obtain a neutralizable antithrombotic, a chimeric molecule (SSR126517E) containing the sequence of a long‐lasting antithrombin (AT)‐dependent anti‐factor Xa pentasaccharide, idraparinux, linked to a biotin molecule was synthesized and tested for anticoagulant and antithrombotic activity. Methods: SSR126517E was tested in several models in vitro and in vivo for its pharmacological properties as well as its ability to be neutralized by avidin. Results: SSR126517E displayed exactly the same properties as idraparinux. In vitro, SSR126517E had a very high affinity for AT (K_d < 1 nm ) and showed a potent anti‐FXa effect and inhibition of thrombin generation with IC₅₀ values similar to those of idraparinux. Ex vivo, after intravenous administration to rats, SSR126517E produced a potent and long‐lasting anti‐FXa effect comparable to that obtained with idraparinux; as with idraparinux, the subcutaneous bioavailability was 100%. In vivo, SSR126517E was a potent antithrombotic in rat and mouse venous and arterial thrombosis models. Direct comparison in rats showed that SSR126517E was as active as idraparinux, when administered at the same molar dose. Furthermore, injection of avidin triggered the immediate elimination of SSR126517E from the bloodstream, resulting in complete neutralization of the antithrombotic activity of SSR126517E. Conclusions: These results show for the first time that coupling an oligosaccharide with biotin has no effect on the former’s pharmacokinetic and pharmacologic properties and renders neutralization easy by injection of avidin.     

Model thrombi formed under flow reveal the role of factor XIII‐mediated cross‐linking in resistance to fibrinolysis

Summary. Background: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin–fibrin and fibrin–inhibitor cross‐links, resulting in more mechanically stable clots. The impact of cross‐linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. Methods: We used a whole blood model thrombus system to characterize the role of cross‐linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. Results: A site‐specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet‐rich head and fibrin‐rich tail. Model thrombi from an FXIII‐deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient’s preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo. Conclusions: Model thrombi formed under flow, even those prepared as plasma ‘thrombi’, reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve γ‐dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis.     

Secretion and antifibrinolytic function of thrombin‐activatable fibrinolysis inhibitor from human platelets

Summary. Background: The thrombin‐activatable fibrinolysis inhibitor (TAFI) is a zymogen first characterized in human plasma that is activated through proteolytic cleavage by thrombin, thrombin in complex with thrombomodulin, or plasmin. Active TAFI attenuates fibrinolysis by removing C‐terminal lysine residues from partially degraded fibrin, thereby inhibiting a potent positive feedback loop in the fibrinolytic cascade. The existence of a separate pool of TAFI within platelets has been described. Objectives and Methods: We aimed to confirm the presence of TAFI in the medium of washed, thrombin‐stimulated platelets and to evaluate the characteristics of platelet TAFI by western blot analysis and with a quantitative assay for activated TAFI. We also assessed the ability of platelet TAFI to inhibit fibrinolysis in vitro, using a platelet‐rich thrombus lysis assay. Results: Our data are consistent with the presence of TAFI in the α‐granules of resting platelets. In contrast to previous reports, platelet TAFI is very similar in electrophoretic mobility to plasma‐derived TAFI. We also show, for the first time, that platelet‐derived TAFI is capable of attenuating platelet‐rich thrombus lysis in vitro independently of plasma TAFI. Moreover, we demonstrate additive effects on thrombolysis of platelet‐derived TAFI and TAFI present in plasma. Conclusions: Taken together, these observations indicate that the secretion of platelet‐derived TAFI can augment the concentrations of TAFI already present in plasma to enhance attenuation of the fibrinolytic cascade. This could be significant in regions of vascular damage or pathologic thrombosis, where activated platelets are known to accumulate.     

A heparin mimetic isolated from a marine shrimp suppresses neovascularization

Summary. Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age‐related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin‐like compound with striking anti‐inflammatory action and negligible anticoagulant and hemorrhagic activities. Objectives: To investigate the role of this novel heparin‐like compound in angiogenic processes. Methods and Results: The anti‐angiogenic effect of this heparinoid in laser‐induced CNV and in vitro models is reported. The compound binds to growth factors (FGF‐2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2‐D network organization in capillary‐like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF‐β1 in the choroidal tissue. The low content of 2‐O‐sulfate groups in this heparinoid may explain its potent anti‐angiogenic effect. Conclusions: The properties of the shrimp heparinoid, such as potent anti‐angiogenic and anti‐inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti‐angiogenic effect of the heparinoid is proposed.     

The profibrotic cytokine transforming growth factor‐β1 increases endothelial progenitor cell angiogenic properties

Summary. Background: Transforming growth factor‐β1 (TGF‐β1) is a profibrotic cytokine that plays a major role in vascular biology, and is known to regulate the phenotype and activity of various vascular cell populations. Because most fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), are associated with vascular remodeling, and as endothelial progenitor cells (EPCs) may be involved in this process, we investigated the impact of TGF‐β1 modulation of EPC angiogenic properties. Methods: TGF‐β1 plasma levels were determined in 64 patients with IPF and compared with those in controls. The effect of TGF‐β1 on angiogenesis was studied in vivo in a Matrigel plug model and in vitro on endothelial colony‐forming cells (ECFCs). We studied the effects of inhibiting the expression of the three main receptors of TGF‐β1 in ECFCs by using short interfering RNA. Results: Total TGF‐β1 plasma levels were significantly increased in patients with IPF as compared with controls (P < 0.0001). TGF‐β1 had proangiogenic effects in vivo by increasing hemoglobin content and blood vessel formation in Matrigel plugs implanted in C57/Bl6 mice, and in vitro by enhancing ECFC viability and migration. The effects were abolished by silencing the three main TGF‐β1 receptors. Conclusions: TGF‐β1 is proangiogenic in vivo and induces ECFC angiogenic properties in vitro, suggesting that TGF‐β1 may play a role during vascular remodeling in fibrotic disease states via EPCs.     

EMMPRIN (CD147/basigin) mediates platelet–monocyte interactions in vivo and augments monocyte recruitment to the vascular wall

Summary. Background: Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. Objectives: To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet–monocyte interactions. Methods and Results: Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet–monocyte interactions under flow conditions. Monocytes readily bound to surface‐adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA‐1 were significantly decreased when ANA‐1 cells were pretreated with EMMPRIN‐silencing small interfering RNA prior to injection into wild‐type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN‐silenced ANA‐1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA‐1–platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN‐mediated platelet–monocyte aggregate formation in vivo. Conclusions: EMMPRIN supports platelet–monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development.     

Heparin‐associated thrombocytopenia in 24 401 patients with venous thromboembolism: findings from the RIETE Registry1

Summary. Background: Whether the treatment of venous thromboembolism (VTE) with unfractionated heparin (UFH) confers a higher risk of thrombocytopenia than does treatment with low molecular weight heparin (LMWH) remains controversial, and very few data are available from routine clinical practice. Objectives: We assessed the incidence, risk factors and prognosis of heparin‐associated thrombocytopenia (HAT) according to the type of heparin therapy, UFH or LMWH. Patients/Methods: Data were obtained from the international prospective Registro Informatizado de la Enfermedad TromboEmbolica venosa (RIETE), which included 25 369 patients with confirmed VTE until February 2009. Among them, 24 401 patients were treated either with UFH or with LMWH, and had available information about the 6‐month occurrence of confirmed thrombocytopenia, defined as a platelet count ≤ 150 000 mm^–3. Results: One hundred and forty‐one patients receiving UFH and/or LMWH developed thrombocytopenia within a 6‐month period. The incidence of HAT was significantly higher in the UFH group (1.36%, 95% confidence interval [CI] 0.79–2.17) than in the LMWH group (0.54%, 95% CI 0.44–0.64). As compared with LMWH, UFH significantly increased the risk of HAT in female patients (adjusted hazard ratio [HR] 4.90%, 95% CI 2.58–9.31, P = 0.001) but not in male patients (adjusted HR 1.60%, 95% CI 0.64–3.97, P = 0.31); P = 0.027 for comparison. In each gender, the UFH‐associated excess risk was confined to patients with VTE unrelated to cancer. The poor prognosis of patients with thrombocytopenia was not influenced by the type of heparin therapy. Conclusions: In routine clinical practice, treatment of VTE with UFH seems to confer a higher risk of thrombocytopenia than does treatment with LMWH, especially in women and non‐cancerous patients.