Adili Reheman

Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo

Summary.  Background: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg‐deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF−/−). Methods and results: Here we studied Fg/VWF‐independent platelet aggregation in vitro and found no aggregation in citrated platelet‐rich plasma of Fg/VWF−/− mice. Surprisingly, in Fg/VWF−/− plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF−/− platelets but not of β3‐integrin‐deficient (β3−/−) platelets. In addition, β3−/− platelets did not significantly incorporate into thrombi in Fg/VWF−/− mice. This Fg/VWF‐independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF‐NH2) induced aggregation of gel‐filtered Fg/VWF−/− platelets in 1 mm Ca2+ PIPES buffer. Notably, aggregation in PIPES buffer was only 50–60% of that observed in Fg/VWF−/− plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF−/− mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca2+in vitro. Conclusions: β3 integrin, thrombin, and Ca2+ play critical roles in this Fg/VWF‐independent aggregation, and both plasma and platelet granule proteins contribute to this process.

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)-gamma-chain. ceacam1(-/-) platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1(-/-) mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1(-/-) mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1(-/-) mice showed a reversal in the more stable thrombus growth phenotype. ceacam1(-/-) mice were more susceptible to type I collagen-induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.

Animal model of fetal and neonatal immune thrombocytopenia: role of neonatal Fc receptor in the pathogenesis and therapy

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder in which maternal antibodies cross the placenta and destroy fetal/neonatal platelets. It has been demonstrated that the neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNIT has not been studied. Here, we developed an animal model of FNIT using combined β3 integrin-deficient and FcRn-deficient (β3(-/-)FcRn(-/-)) mice. We found that β3(-/-)FcRn(-/-) mice are immunoresponsive to β3(+/+)FcRn(-/-) platelets. The generated antibodies were β3 integrin specific and were maintained at levels that efficiently induced thrombocytopenia in adult β3(+/+)FcRn(-/-) mice. FNIT was observed when immunized β3(-/-)FcRn(+/+) females were bred with β3(+/+)FcRn(+/+) males, while no FNIT occurred in β3(-/-)FcRn(-/-) females bred with β3(+/+)FcRn(-/-) males, suggesting that FcRn is indispensable for the induction of FNIT. We further demonstrated that fetal FcRn was responsible for the transplacental transport of various IgG isotypes. We found that anti-FcRn antibody and intravenous IgG prevented FNIT, and that intravenous IgG ameliorated FNIT through both FcRn-dependent and -independent pathways. Our data suggest that targeting FcRn may be a potential therapy for human FNIT as well as other maternal pathogenic antibody-mediated diseases.

Mice with deleted multimerin 1 and α-synuclein genes have impaired platelet adhesion and impaired thrombus formation that is corrected by multimerin 1☆

BACKGROUND Multimerin 1 is a stored platelet and endothelial cell adhesive protein that shows significant conservation. In vitro, multimerin 1 supports platelet adhesion and it also binds to collagen and enhances von Willebrand factor-dependent platelet adhesion to collagen. As selective, multimerin 1 deficient mice have not been generated, we investigated multimerin 1 effects on platelet adhesion using a subpopulation of C57BL/6J mice with tandem deletion of the genes for multimerin 1 and alpha-synuclein, a protein that inhibits alpha-granule release in vitro. We postulated that multimerin 1/alpha-synuclein deficient mice might show impaired platelet adhesive function from multimerin 1 deficiency and increased alpha-granule release from alpha-synuclein deficiency. METHODS Platelet function was assessed by intravital microscopy, after ferric chloride injury, using untreated and human multimerin 1-transfused multimerin 1/alpha-synuclein deficient mice, and by in vitro assays of adhesion, aggregation and thrombin-induced P-selectin release. RESULTS Multimerin 1/alpha-synuclein deficient mice showed impaired platelet adhesion and their defective thrombus formation at sites of vessel injury improved with multimerin 1 transfusion. Although multimerin 1/alpha-synuclein deficient platelets showed increased P-selectin release at low thrombin concentrations, they also showed impaired adhesion to collagen, and attenuated aggregation with thrombin, that improved with added multimerin 1. CONCLUSIONS Our data suggest that multimerin 1 supports platelet adhesive functions and thrombus formation, which will be important to verify by generating and testing selective multimerin 1 deficient mice.

Glucagon-Like Peptide 1 Receptor Activation Attenuates Platelet Aggregation and Thrombosis.

Short-term studies in subjects with diabetes receiving glucagon-like peptide 1 (GLP-1)–targeted therapies have suggested a reduced number of cardiovascular events. The mechanisms underlying this unexpectedly rapid effect are not known. We cloned full-length GLP-1 receptor (GLP-1R) mRNA from a human megakaryocyte cell line (MEG-01), and found expression levels of GLP-1Rs in MEG-01 cells to be higher than those in the human lung but lower than in the human pancreas. Incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in MEG-01 cells, and exenatide significantly inhibited thrombin-, ADP-, and collagen-induced platelet aggregation. Incubation with exenatide also inhibited thrombus formation under flow conditions in ex vivo perfusion chambers using human and mouse whole blood. In a mouse cremaster artery laser injury model, a single intravenous injection of exenatide inhibited thrombus formation in normoglycemic and hyperglycemic mice in vivo. Thrombus formation was greater in mice transplanted with bone marrow lacking a functional GLP-1R (Glp1r−/−), compared with those receiving wild-type bone marrow. Although antithrombotic effects of exenatide were partly lost in mice transplanted with bone marrow from Glp1r−/− mice, they were undetectable in mice with a genetic deficiency of endothelial nitric oxide synthase. The inhibition of platelet function and the prevention of thrombus formation by GLP-1R agonists represent potential mechanisms for reduced atherothrombotic events.

The Cell Motility Modulator Slit2 Is a Potent Inhibitor of Platelet Function

Background— Vascular injury and atherothrombosis involve vessel infiltration by inflammatory leukocytes, migration of medial vascular smooth muscle cells to the intimal layer, and ultimately acute thrombosis. A strategy to simultaneously target these pathological processes has yet to be identified. The secreted protein, Slit2, and its transmembrane receptor, Robo-1, repel neuronal migration in the developing central nervous system. More recently, it has been appreciated that Slit2 impairs chemotaxis of leukocytes and vascular smooth muscle cells toward diverse inflammatory attractants. The effects of Slit2 on platelet function and thrombus formation have never been explored. Methods and Results— We detected Robo-1 expression in human and murine platelets and megakaryocytes and confirmed its presence via immunofluorescence microscopy and flow cytometry. In both static and shear microfluidic assays, Slit2 impaired platelet adhesion and spreading on diverse extracellular matrix substrates by suppressing activation of Akt. Slit2 also prevented platelet activation on exposure to ADP. In in vivo studies, Slit2 prolonged bleeding times in murine tail bleeding assays. Using intravital microscopy, we found that after mesenteric arteriolar and carotid artery injury, Slit2 delayed vessel occlusion time and prevented the stable formation of occlusive arteriolar thrombi. Conclusions— These data demonstrate that Slit2 is a powerful negative regulator of platelet function and thrombus formation. The ability to simultaneously block multiple events in vascular injury may allow Slit2 to effectively prevent and treat thrombotic disorders such as myocardial infarction and stroke.

Integrin PSI domain has endogenous thiol isomerase function and is a novel target for anti-platelet therapy.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

The fibrinogen but not the Factor VIII content of transfused plasma determines its effectiveness at reducing bleeding in coagulopathic mice

The evidence supporting plasma transfusion as a means to restore hemostatic control and prevent or treat bleeding is weak, leading to uncertainties as to which proteins affect the therapeutic quality of plasma. Some regulators focus on coagulation Factor (F)VIII activity, but whether this measure reflects overall transfusable plasma efficacy is questionable. We developed a mouse model of coagulopathy in which bleeding outcomes were responsive to plasma transfusion and addressed the relative contributions of FVIII and fibrinogen (Fg) to plasma quality.

Prevention of surface-induced thrombogenesis on poly(vinyl chloride)

Much biomedical equipment consisting of or containing plastic polymer(s) must come into contact with blood - an interaction that, at the molecular level, may unfortunately prompt biological processes with potentially deleterious, short- or long-term effects such as thrombosis. In the present investigation, this problem is alleviated for poly(vinyl chloride) (PVC) through chemical surface modification with an ultrathin, monoethylene glycol-based coating - a transformation that is characterized using X-ray photoelectron spectroscopy (XPS) supplemented by contact angle goniometry (CAG). Antithrombogenic properties are assessed through calculation (for the first 10 min, and after 60 min) of the surface coverage percentage due to platelet adhesion, aggregation and thrombus formation upon continuous exposure to fluorescently-labelled whole human blood. At all shear rates investigated (300, 900, and 1500 s-1), surface coverage decreases by >99% with respect to bare PVC (10 min, short-term contact with blood). Most importantly, antithrombogenic performance is retained for longer-term exposure experiments (60 min), regardless of applied shear rate as well.